US20100304438A1

US20100304438A1 - Novel beta-glucosidase enzymes

Info

Publication number: US20100304438A1
Application number: US12/790,079
Authority: US
Inventors: Brian R. Scott; Chengsong Liu; James Lavigne; John J. Tomashek
Original assignee: Iogen Energy Corp
Current assignee: Iogen Energy Corp
Priority date: 2009-05-29
Filing date: 2010-05-28
Publication date: 2010-12-02
Also published as: CA2763836A1; WO2010135836A1; US8486683B2; WO2010135836A8

Abstract

Provided are modified beta-glucosidase enzymes, derived from the Trichoderma reesei Cel3A beta-glucosidase, that exhibit improvements in one or more kinetic parameters (K_G, K_G2, k_cat) comprising amino acid substitutions at one or more of positions 43, 101, 260 and 543. Also provided are genetic constructs comprising nucleotide sequences encoding for modified beta-glucosidase enzymes, methods for the production of modified beta-glucosidase enzymes from host strains and the use of the modified beta-glucosidase enzymes in the hydrolysis of cellulose.

Description

FIELD OF THE INVENTION

The present invention relates to modified beta-glucosidases. More specifically, the invention relates to modified beta-glucosidases with improved kinetic parameters for the conversion of cellobiose to glucose. The present invention also relates to genetic constructs comprising nucleotide sequences encoding for modified beta-glucosidases, methods for the production of the modified beta-glucosidases from host strains and the use of the modified beta-glucosidase in industrial applications, including the hydrolysis of cellulose.

BACKGROUND OF THE INVENTION

Lignocellulosic feedstocks are a promising alternative to corn starch for the production of fuel ethanol. These raw materials are widely available, inexpensive and several studies have concluded that cellulosic ethanol generates close to zero greenhouse gas emissions.
However, these feedstocks are not easily broken down into their composite sugar molecules. Recalcitrance of lignocellulose can be partially overcome by physical and/or chemical pretreatment. An example of a chemical pretreatment is steam explosion in the presence of dilute sulfuric acid (U.S. Pat. No. 4,461,648). This process removes most of the hemicellulose, but there is little conversion of the cellulose to glucose. The pretreated material may then be hydrolyzed by cellulase enzymes.
The term cellulase broadly refers to enzymes that catalyze the hydrolysis of the beta-1,4-glucosidic bonds joining individual glucose units in the cellulose polymer. The catalytic mechanism involves the synergistic actions of endoglucanases (E.C. 3.2.1.4), cellobiohydrolases (E.C. 3.2.1.91) and beta-glucosidases (E.C. 3.2.1.21) (Henrissat et al, 1994; Knowles et al., 1987; Lynd et al., 2002; Teeri, 1997; Wood and Garcia-Campayo, 1990; Zhang and Lynd, 2004). Endoglucanases hydrolyze accessible glucosidic bonds in the middle of the cellulose chain, while cellobiohydrolases processively release cellobiose from these chain ends. Beta-glucosidases hydrolyze cellobiose to glucose thus minimizing product inhibition of the cellobiohydrolases and endoglucanases.
Beta-glucosidases are produced by many organisms occurring in all five living kingdoms. Generally these enzymes hydrolyze aryl-beta-glucosides, among which is included cellobiose (gluco-beta-(1,4)-glucoside). Some also catalyze transglycosylation reactions to varying extents.
Filamentous fungi, including Trichoderma ssp., Aspergillus ssp., Hypocrea ssp., Humicola ssp., Neurospora ssp., Orpinomyces ssp., Gibberella ssp., Emericella ssp., Chaetomium ssp., Fusarium ssp., Penicillium ssp., Magnaporthe ssp., Chrysosporium ssp., Myceliophthora ssp., Theilavia ssp., Sporotrichum ssp. and Phanerochaete ssp. are effective producers of cellulase enzymes. Many of these organisms secrete beta-glucosidase enzymes. Trichoderma spp. (Trichoderma longibrachiatum or Trichoderma reesei) secrete small amounts of beta-glucosidase I or Cel3A (Chirico et al., 1987) and likely also secrete two other beta-glucosidases, Cel3B and Cel3E (Foreman et al., 2003).
The enzymatic hydrolysis of pretreated lignocellulosic feedstocks is an inefficient step in the production of cellulosic ethanol and its cost constitutes one of the major barriers to commercial viability. Improving enzymatic activity has been widely regarded as an opportunity for significant cost savings.
Cellobiohydrolases are strongly inhibited by cellobiose and to a lesser degree by glucose. Conversion of cellobiose to glucose is a rate-limiting step in cellulose hydrolysis because filamentous fungi, such as Trichoderma reesei, produce very low levels of beta-glucosidase and beta-glucosidases are highly sensitive to glucose inhibition (Chirico et al., 1987; Berghem et al., 1974). One technique for reducing cellulase inhibition is to increase the amount of beta-glucosidase in the system (U.S. Pat. No. 6,015,703), as cellobiose is more inhibitory to cellulases than glucose (Holtzapple et al., 1990; Teleman et al., 1995). However, over-expressing a beta-glucosidase in an organism such as Trichoderma may reduce the production of other cellulase enzymes and, in turn, may limit the rate of cellulose conversion to cellobiose. In addition, this approach does not specifically address the effect of glucose inhibition on beta-glucosidase activity. A complementary approach would be to use a beta-glucosidase with a higher specific activity which is also less sensitive to glucose inhibition. This enzyme would mitigate cellobiose product inhibition, but do so with lower amounts of beta-glucosidase (relative to the amount of cellulase(s)) and maintain its catalytic efficiency in the presence of high glucose concentrations.
Beta-glucosidases from most fungi have binding constants for cellobiose (K_G2) that range from 0.2-2.0 mM (Chirico et al., 1987; Berghem et al., 1974; Enari et al., 1981; Christakopoulos et al., 1994). These enzymes are highly sensitive to glucose inhibition; K_Gvalues for glucose ranging from 0.6-8.0 mM have been reported for these enzymes. Several microbial beta-glucosidases with higher tolerance to glucose inhibition (K_G>8.0 mM) have been reported (Riou et al., 1998; U.S. Pat. No. 6,087,131; Saha et al., 1996; U.S. Pat. No. 5,747,320; Gueguen et al., 1995; Li et al., 1991; Perez-Pons et al., 1994; Chen et al., 1994; U.S. Pat. No. 6,184,018 B1). However, these enzymes generally have a lower affinity for cellobiose (i.e., higher K_G2values). As a result, the concentration of cellobiose at steady state would be higher using these beta-glucosidases, increasing the degree of cellobiose inhibition on cellulase activity. Therefore, these particular glucose tolerant beta-glucosidase enzymes have limited utility for the production of cellulosic ethanol.
In spite of much research effort, there remains a need for improved beta-glucosidase enzymes in order to generate enzyme mixtures with higher sustained hydrolysis activity on pretreated lignocellulosic feedstock. The absence of such improved beta-glucosidase enzymes represents a large hurdle in the commercialization of cellulose conversion to glucose and other soluble fermentable sugars for the production of ethanol and other products.

SUMMARY OF THE INVENTION

The present invention relates to modified beta-glucosidases. More specifically, the invention relates to modified beta-glucosidases with improved kinetic parameters for the conversion of cellobiose to glucose. Beta-glucosidases of the present invention find utility in industrial processes requiring efficient conversion of cellobiose to glucose in the presence of glucose concentrations that would otherwise inhibit a parental beta-glucosidase.
An embodiment of the invention relates to a modified beta-glucosidase of Trichoderma reesei produced by substitution of the amino acid at one or more of positions 43, 101, 260 and 543 in the beta-glucosidase I or TrCel3A sequence (SEQ ID NO: 1) and comprising an amino acid sequence that is from about 80% to 99.9% to that TrCel3A amino acid sequence defined by SEQ ID NO: 1.
The modified TrCel3A beta-glucosidase may be derived from a parental TrCel3A beta-glucosidase that is otherwise identical to the modified TrCel3A beta-glucosidase and includes the substitution of the naturally occurring amino acid at one or more of positions 43, 101, 260 and 543. For example, the modified TrCel3A beta-glucosidase may contain one or more amino acid substitutions at positions other than at positions 43, 101, 260 and 543, provided that the amino acid sequence of the modified TrCel3A is from about 80% to about 99.9% identical to the TrCel3A amino acid sequence (SEQ ID NO: 1). For example, this invention includes the modified TrCel3A as defined above and further comprising an amino acid substitution at one or more of positions 66, 72, 96, 235, 248 and 369.
The present invention also relates to a modified TrCel3A beta-glucosidase comprising an amino acid sequence that is from about 80% to about 99.9% identical to that of the wild-type TrCel3A of SEQ ID NO: 1 and which exhibits (a) at least a 20% increase in the K_G, (b) at least a 20% decrease in K_G2, or (c) at least a 10% increase in k_catfor cellobiose relative to the K_G, K_G2and/or k_catof a parental TrCel3A beta-glucosidase from which is derived.
The present invention also relates to a modified TrCel3A consisting of the amino acid sequence defined by:

SEQ ID NO: 2 (TrCel3A-V43I);

SEQ ID NO: 3 (TrCel3A-V43C);

SEQ ID NO: 4 (TrCel3A-V101A);

SEQ ID NO: 5 (TrCel3A-V101G);

SEQ ID NO: 6 (TrCel3A-F260I);

SEQ ID NO: 7 (TrCel3A-F260V);

SEQ ID NO: 8 (TrCel3A-F260Q);

SEQ ID NO: 9 (TrCel3A-F260D);

SEQ ID NO: 10 (TrCel3A-I543N);

SEQ ID NO: 11 (TrCel3A-I543W);

SEQ ID NO: 12 (TrCel3A-I543A);

SEQ ID NO: 13 (TrCel3A-I543S);

SEQ ID NO: 14 (TrCel3A-I543G);

SEQ ID NO: 15 (TrCel3A-I543L);

SEQ ID NO: 16 (TrCel3A-572N-V101M-F260I);

SEQ ID NO: 17 (TrCel3A-V43I-572N-V101M);

SEQ ID NO: 18 (TrCel3A-572N-V101M-I543N);

SEQ ID NO: 19 (TrCel3A-572N-V101M-I543D);

SEQ ID NO: 20 (TrCel3A-572N-V101M-I543L);

SEQ ID NO: 21 (TrCel3A-V43I-572N-V101M-F260I);

SEQ ID NO: 22 (TrCel3A-V43I-572N-V101M-F260I-I543N);

SEQ ID NO: 23 (TrCel3A-V43I-572E-V101M-I543N);

SEQ ID NO: 69 (TrCel3A-572N-V101M-F260I-I543N); or

SEQ ID NO: 70 (TrCel3A-572N-V101M-F260I-I543L).

The genetic constructs of the present invention comprise a nucleic acid sequence encoding a modified TrCel3A with an amino acid sequence that is from about 80% to about 99.9% amino acid sequence identity to SEQ ID NO: 1 and that comprises an amino acid substitution at one or more of positions 43, 101, 260 and 543, which nucleic acid sequence is operably linked to nucleic acid sequences regulating its expression and secretion from a host microbe. For example, the nucleic acid sequences regulating the expression and secretion of the modified TrCel3A beta-glucosidase may be derived from the host microbe used for expression of the modified TrCel3A beta-glucosidase. The host microbe may be a yeast, such as Saccharomyces cerevisiae, or a filamentous fungus, such as Trichoderma reesei.
The invention also relates to a genetic construct as defined above, wherein the modified TrCel3A beta-glucosidase comprises an amino acid sequence that is from about 90% to about 99.9% identical to SEQ ID NO: 1. The modified TrCel3A beta-glucosidase may further comprise substitutions at one or more of positions 66, 72, 96, 235, 248, 369 and 386 or any other additional mutations.
The invention also relates to a genetically modified microbe comprising a genetic construct encoding the modified TrCel3A beta-glucosidase. For example, the genetically modified microbe may be capable of expression and secretion of the modified TrCel3A beta-glucosidase further comprising substitution at one or more of positions 66, 72, 96, 235, 248, 369 and 386 or any other additional mutations. The genetically modified microbe may be a yeast or filamentous fungus. For example, the genetically modified microbe may be a species of Saccharomyces, Pichia, Hansenula, Trichoderma, Hyprocrea, Aspergillus, Fusarium, Humicola, Chrysosporium, Myceliophthora, Thielavia, Sporotrichum or Neurospora.
The present invention also relates to the use of the modified TrCel3A beta-glucosidase in a hydrolysis reaction containing a cellulosic substrate and a cellulase mixture comprising the modified TrCel3A beta-glucosidase.
The invention also relates to a process of producing the modified TrCel3A beta-glucosidase as defined above, including providing a yeast or fungal host with a genetic construct comprising a nucleic acid sequence encoding the modified TrCel3A beta-glucosidase, selection of recombinant yeast or fungal strains expressing the modified TrCel3A beta-glucosidase, culturing the selected recombinant strains in submerged liquid fermentations under conditions that induce the expression of the modified TrCel3A beta-glucosidase and recovering the modified TrCel3A beta-glucosidase.
Such modified TrCel3A beta-glucosidases find use in a variety of applications in industrial processes requiring enzymes that can retain high activity in the presence of normally inhibitory concentrations of the glucose. For example, modified TrCel3A beta-glucosidases, as described herein, may be used for the purposes of saccharification of lignocellulosic feedstocks for the production of fermentable sugars or in the production of compounds such as those used in the medical and food industries.
In another embodiment, the invention relates to a modified Family 3 beta-glycosidase comprising one or more of the amino acid substitutions selected from the group consisting of V43I, V43C, V101A, V101G, F260I, F260V, F260Q, F260D, I543N, I543A, I543S, I543G and I543L and having an amino acid sequence that is at least 80% identical to the amino acid sequence of a parental Family 3 beta-glycosidase from which it is derived. The positions of the amino acid substitution(s) are determined from sequence alignment of the Family 3 beta-glycosidase with a Trichoderma reesei Cel3A amino acid sequence as defined in SEQ ID NO: 1. The modified Family 3 beta-glycosidase of the present invention exhibits (a) an increase in the K_p, (b) a decrease in K_sor (c) an increase in k_catrelative to the K_P, K_Sor k_catof the parental Family 3 beta-glycosidase from which it is derived.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts plasmid vector YEp352/PGK91-1/α _ss6H-TrCel3A directing the expression and secretion of native and modified TrCel3A from recombinant Saccharomyces cerevisiae.

FIG. 2 shows a Saccharomyces cerevisiae growth plate containing synthetic complete medium plus 0.1% esculin hydrate and 0.03% FeCl₃. Colonies which turned black after incubation for 3-4 days at 30° C. were identified as expressing active TrCel3A beta-glucosidase.

FIG. 3 is a scatter plot of beta-glucosidase activity in assay 1 (30 mM cellobiose) versus beta-glucosidase activity in assay 2 (5 mM cellobiose+1.25 mM glucose). The data relate to the screening of one 96-well culture plate containing wild-type TrCel3A (wt), filtrates from empty vector transformants (EV) and modified TrCel3As (variants). The wild-type TrCel3A data were fit by linear regression in which the y-intercept was fixed to zero.

FIG. 4 shows an SDS-PAGE gel of the parental (wt) and modified TrCel3A beta-glucosidases expressed from yeast and purified as described in Example 7, as well as a cellulase enzyme mixture from Trichoderma reesei (cellulase) and the wild-type TrCel3A purified from T. reesei cellulase (Cel3A). After SDS-PAGE separation, the proteins were visualized by Coomassie Blue stain.

FIG. 5 is a diagram illustrating the reaction scheme of the pNPG competitive substrate/inhibitor kinetic assay.

FIG. 6 shows the competitive substrate/inhibitor assay results for parental TrCel3A (Wt).

FIG. 7 shows the competitive substrate/inhibitor assay results for the modified TrCel3A-V43I beta-glucosidase.

FIG. 8 shows the competitive substrate/inhibitor assay results for the modified TrCel3A-V101A beta-glucosidase.

FIG. 9 shows the competitive substrate/inhibitor assay results for the modified TrCel3A-F260I beta-glucosidase.

FIG. 10 shows the competitive substrate/inhibitor assay results for the modified TrCel3A-I543N beta-glucosidase.

FIG. 11 shows an alignment of the amino acid sequences of 45 fungal Family 3 beta-glucosidases, including the parental TrCel3A of SEQ ID NO: 1, a consensus Family 3 beta-glucosidase sequence, and the % sequence identity of each amino acid sequence to that of TrCel3A. The positions of V43, V101, F260, and I543 are indicated by asterisks (*); the positions of the catalytic amino acids D236 and E447 are indicated by arrows (↓). A graphical representation of the frequency of occurrence of the amino acid at each position of the consensus Family 3 beta-glucosidase sequence of FIG. 11 among the 45 fungal Family 3 beta-glucosidases is provided below the aligned sequences.

FIG. 12 is a Michaelis-Menton plot comparing the rates of cellobiose hydrolysis by wild-type parental TrCel3A and modified TrCel3A-F260I at different substrate concentrations.

FIG. 13 depicts plasmid vector pc/xCel3A-S72N-V101M-F260I-pyr4-TV directing the expression and secretion of modified TrCel3A beta-glucosidase from genetically modified Trichoderma reesei.

FIG. 14 shows the relative abundance of modified TrCel3A beta-glucosidases in cellulase mixtures produced by genetically modified Trichoderma reesei.

FIG. 15 is a plot showing the conversion of cellulose in a pretreated lignocellulosic substrate by cellulase mixtures comprising parental (WT TrCel3A) or modified (Modified TrCel3A) beta-glucosidases produced by genetically modified Trichoderma reesei.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to modified beta-glucosidases. More specifically, the invention relates to modified beta-glucosidases with one or more of an increased K_G, a decreased K_G2, and an increase in k_cat. The present invention also relates to genetic constructs comprising nucleotide sequences encoding for the modified beta-glucosidase, methods for the production of the modified beta-glucosidase from host strains and the use of the modified beta-glucosidase in the hydrolysis of cellulosic substrates, such as pretreated lignocellulosic feedstocks.
The following description is of a preferred embodiment by way of example only and without limitation to the combination of features necessary for carrying the invention into effect. The headings provided are not meant to be limiting of the various embodiments of the invention. Terms such as “comprises”, “comprising”, “comprise”, “includes”, “including” and “include” are not meant to be limiting. In addition, the use of the singular includes the plural, and “or” means “and/or” unless otherwise stated. Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

Modified Beta-Glucosidases

The term “beta-glucosidase” is intended as described by Bhatia et al. (2002). Beta-glucosidase (3.2.1.21) enzymes transfer a glycosyl group between oxygen nucleophiles, generally resulting in the hydrolysis of a beta-glucosidic bond linking carbohydrate residues in aryl-, amino-, alkyl-beta-D-glucosides, cyanogenic-glucosides, short chain oligosaccharides and disaccharides. In oligosaccharides containing more than two glucosides, beta-glucosidase activity decreases as chain length increases. Beta-glucosidases hydrolyze beta-1,4-glucosidic bonds via a double displacement reaction, resulting in a net retention of anomeric configuration. Two acidic amino acids, aspartic (D) and/or glutamic (E) acid, are directly involved in substrate catalysis. One of these residues acts as a nucleophile and forms an enzyme-glycosyl intermediate. The other acidic residue acts as an acid-base catalyst. In the Trichoderma reesei beta-glucosidase 1, herein referred to as TrCel3A whose amino acid sequence is presented in SEQ ID NO: 1, the asparagine at position 236 serves as the nucleophile and the glutamine at position 447 is the acid-base catalyst.
Beta-glucosidases are a subset of beta-glycosidases belonging to glycoside hydrolase (GH) Families 1 and 3, using the classification system developed by Henrissat and coworkers (Henrissat, B. (1991); Henrissat, B. and Bairoch, A. (1996)). There are currently over 115 GH families that have been identified using this classification system, which are listed in the database of Carbohydrate Active Enzymes (CAZy) (see http://afmb.cnrs-mrs.fr/CAZY/index.html for reference). Family 1 comprises beta-glycosidases from archaebacteria, plants and animals. Beta-glycosidases from some bacteria, mold and yeast belong to Family 3. For the purpose of this invention, a “beta-glycosidase” is therefore defined as any protein that is categorized as a Family 3 glycoside hydrolase according to the CAZy system.
The three dimensional structure of beta-D-glucan exohydrolase, a Family 3 glycosyl hydrolase, was described by Varghese et al. (1999). The structure was of a two domain globular protein comprising a N-terminal (α/β)₈TIM-barrel domain and a C-terminal a six-stranded beta-sandwich, which contains a beta-sheet of five parallel beta-strands and one antiparallel beta-strand, with three alpha-helices on either side of the sheet. This structure is likely shared by other Family 3 enzymes.
As shown in FIG. 11, the primary amino acid sequence of Family 3 beta-glucosidases show a high degree of similarity. Multiple alignment across 45 Family 3 beta-glucosidase amino acid sequences shows that the most naturally occurring Family 3 beta-glucosidases of fungal origin show from about 40% to about 100% amino acid sequence identity to the amino acid sequence of TrCel3A (FIG. 11). In particular, there are several regions of very high amino acid sequence conservation within the Family 3 beta-glucosidases including, for example, from amino acids 225-256 and 439-459, containing the catalytic amino acids D236 and E447, respectively.
By “TrCel3A beta-glucosidase” or “TrCel3A” it is meant the Family 3 glycosyl hydrolase produced by Trichoderma reesei defined by the amino acid sequence of SEQ ID NO: 1. TrCel3A beta-glucosidase is also known as Trichoderma reesei beta-glucosidase or BGL1. By “native” or “wild type” TrCel3A (also annotated as TrCel3A^wt), it is meant the TrCel3A of SEQ ID NO: 1 without any amino acid substitutions.
By “modified TrCel3A beta-glucosidase” or “modified TrCel3A”, it is meant a TrCel3A beta-glucosidase which comprises one or more of the amino acid substitutions, introduced by genetic engineering techniques, selected from the group consisting of V43X, V101X, F260X, and I543X. For example, the modified TrCel3A beta-glucosidase may comprising one or more amino acid substitutions selected from the group consisting of V43I V43C, V101A, V101G, F260I, F260V, F260Q, F260D, I543N, I543A, I543S, I543G and I543L.
Genetic engineering techniques for altering amino acid sequences include, but are not limited to, site-directed mutagenesis, cassette mutagenesis, random mutagenesis, synthetic oligonucleotide construction, cloning and other genetic engineering techniques as would be known by those of skill in the art (Eijsink V G, et al. 2005). Modified TrCel3A beta-glucosidases of the present invention include those comprising amino acid substitutions at any one of V43X, V101X, F260X and I543X, at any two of V43X, V101X, F260X and I543X, any three of V43X, V101X, F260X and I543X, or all four of V43X, V101X, F260X and I543X.
It will be understood that the modified TrCel3A beta-glucosidase may be derived from wild-type TrCel3A beta-glucosidase of SEQ ID NO: 1 or from a TrCel3A beta-glucosidase that contains other amino acid substitutions. For example, the modified TrCel3A beta-glucosidase may contain amino acid substitution at one or more of positions 66, 73, 96, 235, 248, and 369. Alternatively, after production of the modified TrCel3A beta-glucosidase comprising mutations at one or more of positions 43, 101, 260 and 543, it may be subsequently further modified to contain additional amino acid substitutions, including but not limited to those set forth above.
As used herein in respect of modified TrCel3A beta-glucosidase amino acid sequences, “derived from” refers to the isolation of a target nucleic acid sequence element encoding the desired modified TrCel3A beta-glucosidase using genetic material or nucleic acid or amino acid sequence information specific to the parental TrCel3A beta-glucosidase. As is known by one of skill in the art, such material or sequence information can be used to generate a nucleic acid sequence encoding the desired modified TrCel3A beta-glucosidase using one or more molecular biology techniques including, but not limited to, cloning, sub-cloning, amplification by PCR, in vitro synthesis, and the like.
In one embodiment of the invention, the amino acid sequence of the modified TrCel3A beta-glucosidase is from about 80% to about 99.9% identical to SEQ ID NO: 1. For example, the amino acid sequence of the modified TrCel3A beta-glucosidase may be from about 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 99.9% identical to SEQ ID NO: 1. In other words, the number of amino acid substitutions in the modified TrCel3A beta-glucosidase may not exceed 20% of the total number amino acids in the parental TrCel3A beta-glucosidase sequence.
In another embodiment of the invention, the amino acid sequence of modified TrCel3A beta-glucosidase may be from about 90% to about 99.9% identical to SEQ ID NO: 1. For example, the amino acid sequence of the modified TrCel3A beta-glucosidase may be from about 95% to about 100% identical to SEQ ID NO: 1.
In another embodiment, the amino acid sequence of the modified TrCel3A beta-glucosidase may be from about 80% to about 99.9% identical to SEQ ID NO: 1 and the modified TrCel3A beta-glucosidase may exhibit (a) at least about a 20% increase in K_G, (b) at least about a 20% decrease in K_G2, or (c) at least about a 10% increase in k_catfor cellobiose relative to the K_G, K_G2and/or k_catof a parental TrCel3A beta-glucosidase from which it is derived. For example, the modified TrCel3A beta-glucosidase may exhibit (a) from about a 20% to about a 800% increase in K_G, or any increase therebetween, (b) from about a 20% to about an 80% decrease in K_G2, any decrease therebetween, or (c) from about a 10% to about a 50% increase in k_catfor cellobiose relative to the K_G, K_G2and/or k_catof a parental TrCel3A beta-glucosidase from which is derived.
By “parental TrCel3A beta-glucosidase” or “parental TrCel3A”, it is meant a TrCel3A beta-glucosidase that does not contain a substitution of its original amino acid(s) at positions 43, 101, 260 or 543. For example, the parental TrCel3A beta-glucosidase may comprise amino acid substitutions at one or more of positions 66, 72, 96, 235, 248, and 369.
In order to assist one of skill in the art regarding those amino acid positions of the TrCel3A beta-glucosidase at which amino acid substitutions (other than V43X, V101X, F260X, and I543X) may be made and produce an active beta-glucosidase, an alignment of 45 Family 3 beta-glucosidases derived from fungal sources along with a consensus beta-glucosidase sequence consisting of the amino acids that naturally occur with the highest frequency at each position is provided in FIG. 11 along with a graph showing the frequency of occurrence of each amino acid of the consensus sequence at each position. Using the information provided in FIG. 11, one of skill in the art would recognize regions of low sequence conservation to other Family 3 beta-glucosidases and choose such regions for introduction of amino acid substitutions that are not likely to compromise significantly the function of the enzyme. Non-limiting examples of such regions include, for example, the regions between positions 1-20, 303-323 and 403-414 and select amino acid positions within these regions.
As described in more detail herein, several modified TrCel3A beta-glucosidases have been prepared that exhibit (a) at least a 20% increase in K_G, (b) at least a 20% decrease in K_G2, or (c) at least a 10% increase in k_catfor cellobiose relative to the K_G, K_G2and/or k_catof a parental TrCel3A beta-glucosidase from which is derived. A list of several modified TrCel3A beta-glucosidases, which is not to be considered limiting in any manner, is presented in Table 1.

TABLE 1

TrCel3A beta-glucosidases with improved catalytic efficiency

	Modified TrCel3A beta-glucosidase	SEQ ID NO:

	TrCel3A-V43I	2
	TrCel3A-V43C	3
	TrCel3A-V101A	4
	TrCel3A-V101G	5
	TrCel3A-F260I	6
	TrCel3A-F260V	7
	TrCel3A-F260Q	8
	TrCel3A-F260D	9
	TrCel3A-I543N	10
	TrCel3A-I543W	11
	TrCel3A-I543A	12
	TrCel3A-I543S	13
	TrCel3A-I543G	14
	TrCel3A-I543L	15
	TrCel3A-S72N-V101M-F260I	16
	TrCel3A--V43I-S72N-V101M	17
	TrCel3A-S72N-V101M-I543N	18
	TrCel3A-S72N-V101M-I543D	19
	TrCel3A-S72N-V101M-I543L	20
	TrCel3A-V43I-S72N-V101M-F260I	21
	TrCel3A-V43I-S72N-V101M-F260I-I543N	22
	TrCel3A-V43I-S72N-V101M-I543N	23
	TrCel3A-S72N-V101M-F260I-I543N	69
	TrCel3A-S72N-V101M-F260I-I543L	70

Modified TrCel3A Beta-Glucosidases Improved Kinetic Parameters

The modified TrCel3A beta-glucosidases of the present invention exhibit improvements in at least one of the following kinetic parameters: K_G, K_G2and k_cat. K_Gis defined as the concentration of glucose which reduces the enzymatic activity of the beta-glucosidase by 50%. K_G2is defined as the concentration of cellobiose at which the beta-glucosidase exhibits half its maximal rate. The k_catis the catalytic rate constant for the hydrolysis of cellobiose. Example 8 details an assay for measuring the K_Gand K_G2of parental and modified TrCel3A beta-glucosidases. Example 9 details an assay for measuring the k_catof parental and modified TrCel3A beta-glucosidases.
K_Gof the parental and modified TrCel3A beta-glucosidases can be determined by measuring the rate of hydrolysis of a chromogenic substrate, such as p-nitrophenyl-beta-D-glucopyranoside (pNPG), in the presence of various concentrations of glucose as described in Example 8. The K_Gis the concentration of glucose that reduces the rate of p-nitrophenol (pNP) release from pNPG by 50% compared to the rate of pNPG hydrolysis in the absence of glucose. The K_G2constants for parental and modified TrCel3A beta-glucosidases can be determined by measuring the rate of hydrolysis of cellobiose in reactions containing increasing concentrations of cellobiose or, alternatively, by measuring the rate of hydrolysis of a chromogenic substrate, such as pNPG, in the presence of various concentrations of a cellobiose as described in Example 8. The K_G2is the concentration of cellobiose that reduces the rate of pNP release from pNPG by 50% compared to the rate of pNPG hydrolysis in the absence of cellobiose. The k_catvalues for parental and modified TrCel3A beta-glucosidases can be determined by measuring the rate of cellobiose hydrolysis at varying concentrations of a cellobiose substrate, for example, as described in Example 9.
The effect of amino acid substitutions at positions 43, 101, 260 and 543 were determined by a comparative study of the modified and parental TrCel3A beta-glucosidases. The relative values of K_G, K_G2and k_catfor the parental and modified TrCel3A beta-glucosidases are shown in Table 2, below. Reaction curves for the hydrolysis of pNPG substrate alone and in the presence of glucose or cellobiose by parental and modified TrCel3A beta-glucosidases are shown in FIGS. 6 through 10. Reaction curves for the hydrolysis of cellobiose substrate by parental and modified TrCel3A beta-glucosidases are shown in FIG. 12.

TABLE 2

Modified TrCel3A Beta-glucosidases with Improved Kinetic Parameters

	Relative	Relative	Relative	Relative
Amino acid substitution	K_G	K_G2	K_G/K_G2	k_cat

None (TrCel3A)	1.00	1.00	1	1
V43I	0.72	0.53	1.38	0.75
V43C	7.24	2.03	3.58	ND
V101A	0.93	0.69	1.34	0.81
F260I	1.59	1.04	1.52	1.24
F260D	1.21	1.11	1.09	ND
F260Q	1.19	1.14	1.05	ND
F260V	1.24	1.08	1.14	ND
I543N	1.50	1.18	1.27	0.73
I543W	0.97	0.81	1.20	0.51
I543S	1.09	0.92	1.19	1.09
I543A	1.17	0.98	1.19	1.00
I543G	0.93	0.86	1.08	0.90
I543L	1.05	0.96	1.10	1.29
S72N-V101M-F260I	0.76	0.58	1.32	0.95
V43I-S72N-V101M	0.59	0.39	1.50	0.85
S72N-V101M-I543N	0.74	0.54	1.37	0.87
S72N-V101M-I543D	0.62	0.45	1.38	ND
S72N-V101M-I543L	0.72	0.58	1.24	0.96
V43I-S72N-V101M-F260I	0.59	0.31	1.92	0.78
S72N-V101M-F260I-I543N	1.10	0.71	1.55	1.02
V43I-S72N-V101M-F260I-I543N	0.71	0.32	2.20	0.69
V43I-S72N-V101M-I543N	0.52	0.27	1.91	0.73
S72N-V101M-F260I-I543L	0.86	0.56	1.54	1.00

Genetic Constructs Encoding Modified TrCel3A Beta-Glucosidases

The present invention also relates to genetic constructs comprising a nucleic acid sequence encoding the modified TrCel3A beta-glucosidase operably linked to regulatory nucleic acid sequences directing the expression and secretion of the modified TrCel3A beta-glucosidase from a host microbe. By “regulatory nucleic acid sequences” it is meant nucleic acid sequences directing the transcription and translation of the modified TrCel3A-encoding nucleic acid sequence and a nucleic acid sequence encoding a secretion signal peptide capable of directing the secretion of the modified TrCel3A beta-glucosidase from a host microbe. The regulatory nucleic acid sequences may be derived from genes that are highly expressed and secreted in the host microbe under industrial fermentation conditions. For example, the regulatory nucleic acid sequences may be derived from any one or more of the Trichoderma reesei cellulase or hemicellulase genes.
The genetic construct may further comprise a selectable marker gene to enable isolation of a genetically modified microbe transformed with the construct as is commonly known to those of skill in the art. The selectable marker gene may confer resistance to an antibiotic or the ability to grow on medium lacking a specific nutrient to the host organism that otherwise could not grow under these conditions. The present invention is not limited by the choice of selectable marker gene, and one of skill in the art may readily determine an appropriate gene. For example, the selectable marker gene may confer resistance to hygromycin, phleomycin, kanamycin, geneticin, or G418, or may complement a deficiency of the host microbe in one of the trp, arg, leu, pyr4, pyr, ura3, ura5, his, or ade genes or may confer the ability to grow on acetamide as a sole nitrogen source.
The genetic construct may further comprise other nucleic acid sequences as is commonly known to those of skill in the art, for example, transcriptional terminators, nucleic acid sequences encoding peptide tags, synthetic sequences to link the various other nucleic acid sequences together, origins of replication, and the like. The practice of the present invention is not limited by the presence of any one or more of these other nucleic acid sequences.

Genetically Modified Microbes Expressing Modified TrCel3A Beta-Glucosidases

The modified TrCel3A beta-glucosidase may be expressed and secreted from a genetically modified microbe produced by transformation of a host microbe with a genetic construct encoding the modified TrCel3A beta-glucosidase. The host microbe may be a yeast or a filamentous fungus, particularly those classified as Ascomycota. Genera of yeasts useful as host microbes for the expression of modified TrCel3A beta-glucosidases of the present invention include Saccharomyces, Pichia, Hansenula, Kluyveromyces, Yarrowia, and Arxula. Genera of fungi useful as microbes for the expression of modified TrCel3A beta-glucosidases of the present invention include Trichoderma, Hypocrea, Aspergillus, Fusarium, Humicola, Neurospora, Chrysosporium, Myceliophthora, Thielavia, Sporotrichum and Penicillium. For example, the host microbe may be an industrial strain of Trichoderma reesei. Typically, the host microbe is one which does not express a parental TrCel3A beta-glucosidase.
The genetic construct may be introduced into the host microbe by any number of methods known by one skilled in the art of microbial transformation, including but not limited to, treatment of cells with CaCl₂, electroporation, biolistic bombardment, PEG-mediated fusion of protoplasts (e.g. White et al., WO 2005/093072, which is incorporated herein by reference). After selecting the recombinant fungal strains expressing the modified TrCel3A, the selected recombinant strains may be cultured in submerged liquid fermentations under conditions that induce the expression of the modified TrCel3A.

Production of Modified TrCel3A Beta-Glucosidases

The modified TrCel3A beta-glucosidase of the present invention may be produced in a fermentation process in which a genetically modified microbe comprising a genetic construct encoding the modified TrCel3A beta-glucosidase is grown in submerged liquid culture fermentation.
Submerged liquid fermentations of microorganisms, including Trichoderma and related filamentous fungi, are typically conducted as a batch, fed-batch or continuous process. In a batch process, all the necessary materials, with the exception of oxygen for aerobic processes, are placed in a reactor at the start of the operation and the fermentation is allowed to proceed until completion, at which point the product is harvested. A batch process for producing the modified TrCel3A beta-glucosidase of the present invention may be carried out in a shake-flask or a bioreactor.
In a fed-batch process, the culture is fed continuously or sequentially with one or more media components without the removal of the culture fluid. In a continuous process, fresh medium is supplied and culture fluid is removed continuously at volumetrically equal rates to maintain the culture at a steady growth rate.
One of skill in the art is aware that fermentation medium comprises a carbon source, a nitrogen source as well as other nutrients, vitamins and minerals which can be added to the fermentation media to improve growth and enzyme production of the host cell. These other media components may be added prior to, simultaneously with or after inoculation of the culture with the host cell.
For the process for producing the modified TrCel3A beta-glucosidase of the present invention, the carbon source may comprise a carbohydrate that will induce the expression of the modified TrCel3A beta-glucosidase from a genetic construct in the genetically modified microbe. For example, if the genetically modified microbe is a strain of a cellulolytic fungus such as Trichoderma, the carbon source may comprise one or more of cellulose, cellobiose, sophorose, xylan, xylose, xylobiose and related oligo- or poly-saccharides known to induce expression of cellulases and beta-glucosidase in such cellulolytic fungi.
In the case of batch fermentation, the carbon source may be added to the fermentation medium prior to or simultaneously with inoculation. In the cases of fed-batch or continuous operations, the carbon source may also be supplied continuously or intermittently during the fermentation process. For example, when the genetically modified microbe is a strain of Trichoderma, the carbon feed rate is between 0.2 and 2.5 g carbon/L of culture/h, or any amount therebetween.
The process for producing the modified TrCel3A beta-glucosidase of the present invention may be carried at a temperature from about 20° C. to about 40° C., or any temperature therebetween, for example from about 25° C. to about 37° C., or any temperature therebetween, or from 20, 22, 25, 26, 27, 28, 29, 30, 32, 35, 37, 40° C. or any temperature therebetween.
The process for producing the modified TrCel3A beta-glucosidase of the present invention may be carried out at a pH from about 3.0 to 6.5, or any pH therebetween, for example from about pH 3.5 to pH 5.5, or any pH therebetween, for example from about pH 3.0, 3.2, 3.4, 3.5, 3.7, 3.8, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.2, 5.4, 5.5, 5.7, 5.8, 6.0, 6.2, 6.5 or any pH therebetween.
Following fermentation, the fermentation broth containing the modified TrCel3A beta-glucosidase may be used directly, or the modified TrCel3A beta-glucosidase may be separated from the fungal cells, for example by filtration or centrifugation. Low molecular solutes such as unconsumed components of the fermentation medium may be removed by ultrafiltration. The modified TrCel3A beta-glucosidase may be concentrated, for example, by evaporation, precipitation, sedimentation or filtration. Chemicals such as glycerol, sucrose, sorbitol and the like may be added to stabilize the modified TrCel3A beta-glucosidase. Other chemicals, such as sodium benzoate or potassium sorbate, may be added to the modified TrCel3A beta-glucosidase to prevent growth of microbial contamination.

The Use of Modified TrCel3A Beta-Glucosidases

The modified TrCel3A beta-glucosidase of the present invention may be used in the hydrolysis of cellulose or in the production of compounds such as those used in the medical and food industries
For use in the enzymatic hydrolysis of cellulose, such as in the production of fermentable sugars from a pretreated lignocellulosic feedstock, the modified TrCel3A beta-glucosidase of the invention may be combined with one or more cellulases to produce a cellulase mixture. In one embodiment of the invention, the modified TrCel3A beta-glucosidase is one of many proteins expressed from a host cell, including, but not limited to, cellulase enzymes. The one or more cellulases in the cellulase enzyme mixture and the modified TrCel3A beta-glucosidase may be secreted from a single genetically modified microbe or by different microbes in combined or separate fermentations. Similarly, the one or more cellulases in the cellulase enzymes mixture with which the modified TrCel3A beta-glucosidase may be combined may be expressed individually or in sub-groups from different strains of different organisms and the enzymes combined to make the cellulase enzyme mixture. It is also contemplated that the enzyme mixture may be expressed individually or in sub-groups from different strains of a single organism, such as from different strains of Saccharomyces, Pichia, Hansenula Trichoderma, Hyprocrea, Aspergillus, Fusarium, Humicola, Chrysosporium, Myceliophthora, Thielavia, Sporotrichum or Neurospora, and the enzymes combined to make the cellulase enzyme mixture. Preferably, all of the enzymes are expressed from a single host organism, such as a strain of cellulolytic fungus belonging to a species of Trichoderma, Hyprocrea, Aspergillus, Fusarium, Humicola, Chrysosporium, Myceliophthora, Thielavia, Sporotrichum or Neurospora.
It is further contemplated that the cellulase mixture may comprise two or more of such modified beta-glucosidases as described here in, each with a unique set of improved kinetic parameters. Such a cellulase mixture would be expected to maintain a constant beta-glucosidase activity across a broad range of conditions. For example, a cellulase mixture may comprise one modified TrCel3A beta-glucosidase with low substrate affinity and low product inhibition (i.e., higher values of K_G2and K_Gthan the parental beta-glucosidase) and one modified TrCel3A beta-glucosidase with high substrate affinity and high product inhibition (i.e., lower values of K_G2and K_Gthan the parental beta-glucosidase). Such a cellulase mixture would exhibit a near level apparent beta-glucosidase activity across a wide range of cellobiose and glucose concentrations. Many possible combinations of two or more beta-glucosidase enzymes might be envisioned to maintain a constant activity across a variety of conditions that could occur across many different processes and applications.
A pretreated lignocellulosic feedstock is a material of plant origin that, prior to pretreatment, contains at least 20% cellulose (dry weight), more preferably greater than about 30% cellulose, even more preferably greater than 40% cellulose, for example 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 55, 60, 65, 70, 75, 80, 85, 90% or any percent therebetween, and at least 10% lignin (dry wt), more typically at least 12% (dry wt) and that has been subjected to physical and/or chemical processes to make the fiber more accessible and/or receptive to the actions of cellulolytic enzymes. After pretreatment, the lignocellulosic feedstock may contain higher levels of cellulose. For example, if acid pretreatment is employed, the hemicellulose component is hydrolyzed, which increases the relative level of cellulose. In this case, the pretreated feedstock may contain greater than about 20% cellulose and greater than about 12% lignin.
Lignocellulosic feedstocks that may be used in the invention include, but are not limited to, agricultural residues such as corn stover, wheat straw, barley straw, rice straw, oat straw, canola straw, sugarcane straw and soybean stover; fiber process residues such as corn fiber, sugar beet pulp, pulp mill fines and rejects or sugar cane bagasse; forestry residues such as aspen wood, other hardwoods, softwood, and sawdust; or grasses such as switch grass, miscanthus, cord grass, and reed canary grass. The lignocellulosic feedstock may be first subjected to size reduction by methods including, but not limited to, milling, grinding, agitation, shredding, compression/expansion, or other types of mechanical action. Size reduction by mechanical action can be performed by any type of equipment adapted for the purpose, for example, but not limited to, a hammer mill.
The enzymatic hydrolysis of cellulose using a cellulase enzyme mixture, as defined above, comprising the modified TrCel3A beta-glucosidase may be batch hydrolysis, continuous hydrolysis, or a combination thereof. The hydrolysis may be agitated, unmixed, or a combination thereof.
The enzymatic hydrolysis may be carried out at a temperature of about 30° C. to about 80° C., or any temperature therebetween, for example a temperature of 30, 35, 40, 45, 50, 55, 60, 65, 70, 75° C., 80° C. or any temperature therebetween, and a pH of about 3.0 to about 8.0, or any pH therebetween, for example at a pH of 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0 or pH therebetween. The initial concentration of cellulose in the hydrolysis reactor, prior to the start of hydrolysis, is preferably about 2% (w/w) to about 15% (w/w), or any amount therebetween, for example 2, 4, 6, 8, 10, 12, 14, 15% or any amount therebetween.
The dosage of the cellulase enzyme mixture comprising the modified TrCel3A beta-glucosidase may be about 0.1 to about 100 mg protein per gram cellulose, or any amount therebetween, for example 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100 mg protein per gram cellulose or any amount therebetween. The hydrolysis may be carried out for a time period of about 1 hours to about 200 hours, or any time therebetween; for example, the hydrolysis may be carried out for a period of 15 hours to 100 hours, or any time therebetween, or it may be carried out for 1, 2, 4, 8, 12, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 120, 140, 160, 180, 200 or any time therebetween. It should be appreciated that the reaction conditions are not meant to limit the invention in any manner and may be adjusted as desired by those of skill in the art.
In practice, the enzymatic hydrolysis is typically carried out in a hydrolysis reactor. The enzyme mixture is added to the pretreated lignocellulosic feedstock (also referred to as the “substrate”) prior to, during, or after the addition of the substrate to the hydrolysis reactor.

Modified Family 3 Beta-Glycosidases

Beta-glucosidases are just one or several classes of hydrolytic enzymes belong to glycoside hydrolase Family 3. For example, Family 3 includes other enzymes that catalyse the hydrolysis of beta-glycosidic bonds such as xylan 1,4-beta-xylosidase (EC 3.2.1.37), beta-N-acetylhexosaminidase (EC 3.2.1.52), glucan 1,3-beta-glucosidase (EC 3.2.1.58), and glucan 1,4-beta-glucosidase (EC 3.2.1.74). For the purposes of the present invention, a “Family 3 beta-glycosidase” is any xylan 1,4-beta-xylosidase (EC 3.2.1.37), beta-N-acetylhexosaminidase (EC 3.2.1.52), glucan 1,3-beta-glucosidase (EC 3.2.1.58), and glucan 1,4-beta-glucosidase (EC 3.2.1.74) that is classified as a Family 3 glycoside hydrolase under the CAZy system (see URL afmb.cnrs-mrs.fr/CAZY/index.html for reference).
By “modified Family 3 beta-glycosidase”, it is meant a Family 3 beta-glycosidase which comprises one or more of the amino acid substitutions, introduced by genetic engineering techniques, selected from the group consisting of V43I, V43C, V101A, V101G, F260I, F260V, F260Q, F260D, I543N, I543A, I543S, I543G and I543L (TrCel3A numbering) and which amino acid sequence is at least 80% identical to the amino acid sequence of the parental Family 3 beta-glycosidase from which it is derived For example, the amino acid sequence of the modified Family 3 beta-glycosidase may be from about 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 99.9% identical to the amino acid sequence of the parental Family 3 beta-glycosidase from which it is derived.
Sequence identity can be readily determined by alignment of the amino acids of the two sequences, either using manual alignment, or any sequence alignment algorithm as known to one of skill in the art, for example but not limited to, BLAST algorithm (BLAST and BLAST 2.0; Altschul et al., 1997 and 1990), the algorithm disclosed by Smith & Waterman (1981), by the homology alignment algorithm of Needleman & Wunsch (1970), by the search for similarity method of Pearson & Lipman (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection. An alignment of 45 Family 3 beta-glycosidase sequences is provided in FIG. 11.
It will be understood that the modified Family 3 beta-glycosidase may be derived from wild-type Family 3 beta-glycosidase or from a Family 3 beta-glycosidase that contains other amino acid substitutions. Alternatively, after production of the modified Family 3 beta-glycosidase comprising mutations selected from the group consisting of V43I, V43C, V101A, V101G, F260I, F260V, F260Q, F260D, I543N, I543A, I543S, I543G and I543L, it may be subsequently further modified to contain additional amino acid substitutions, including but not limited to those set forth above.
By “TrCel3A numbering” it is meant the numbering corresponding to the position of amino acids based on the amino acid sequence of TrCel3A (SEQ ID NO:1) based on alignment of the amino acid sequence of the Family 3 beta-glycosidase with the TrCel3A amino acid sequence. An example of the alignment of 44 other Family 3 beta-glycosidase amino acid sequences with the TrCel3A beta-glucosidase amino acid sequence is provided in FIG. 11.
As used herein in respect of modified Family 3 beta-glycosidase amino acid sequences, “derived from” refers to the isolation of a target nucleic acid sequence element encoding the desired modified Family 3 beta-glycosidase using genetic material or nucleic acid or amino acid sequence information specific to the corresponding parental Family 3 beta-glycosidase. As is known by one of skill in the art, such material or sequence information can be used to generate a nucleic acid sequence encoding the desired modified Family 3 beta-glycosidase using one or more molecular biology techniques including, but not limited to, cloning, sub-cloning, amplification by PCR, in vitro synthesis, and the like.
In one embodiment of the invention of the invention, the amino acid sequence of the modified Family 3 beta-glycosidase is from about 80% to about 99.9% identical to the amino acid sequence of the parental Family 3 beta-glycosidase from which it is derived. For example, the amino acid sequence of the Family 3 beta-glycosidase may be from about 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 99.9% identical to the amino acid sequence of the parental Family 3 beta-glycosidase from which it is derived. In other words, the number of amino acid substitutions in the modified Family 3 beta-glycosidase does not exceed 20% of the total number amino acids in the parental Family 3 beta-glycosidase sequence.
The modified Family 3 beta-glycosidase of the present invention exhibit improvements in at least on of the following kinetic parameters: K_P, K_Sand k_cat. K_Pis defined as the concentration of product which reduces the enzymatic activity of the Family 3 beta-glycosidase by 50%. K_Sis defined as the concentration of substrate at which the Family 3 beta-glycosidase exhibits half its maximal rate. The k_catis the catalytic rate constant for the hydrolysis of substrate.
In another embodiment of the invention, the amino acid sequence of the modified Family 3 beta-glycosidase is from about 90% to about 99.9% identical to the amino acid sequence of the parental Family 3 beta-glycosidase from which it is derived. For example, the amino acid sequence of the modified Family 3 beta-glycosidase may be from about 95% to about 100% identical to SEQ ID NO: 1.
In another embodiment, the amino acid sequence of the modified Family 3 beta-glycosidase may be from about 80% to about 99.9% identical to the amino acid sequence of the parental Family 3 beta-glycosidase from which it is derived, and the modified Family 3 beta-glycosidase may exhibit (a) an increase in the K_P, (b) a decrease in K_S, or (c) an increase in k_catrelative to the K_P, K_Sand/or k_catof a parental Family 3 beta-glycosidase from which is derived.
By “parental Family 3 beta-glycosidase”, it is meant a Family 3 beta-glycosidase that does not contain:
isoleucine or cysteine at position 43,
alanine or glycine at position 101;
isoleucine, valine, glutamine, aspartic acid at position 260; or
asparagine, alanine, serine, glycine or leucine at position 543.
The modified Family 3 beta-glycosidase may be derived from a parental Family 3 beta-glycosidase that comprises one or more naturally-occurring amino acid(s) at the substituted positions corresponding to that of the modified Family 3 beta-glycosidase, but that is otherwise identical to the modified Family 3 beta-glycosidase, for example a native Family 3 beta-glycosidase from A. nidulans—AN1804.2, B. fuckeliana, T. aurantiacus levisporus. The parental Family 3 beta-glycosidase may contain one or more amino acid substitutions at other positions, given that these substitutions are also present in the corresponding modified Family 3 beta-glycosidase. Family 3 beta-glycosidases suitable as parental beta-glycosidases from which modified Family 3 beta-glycosidases may be derived are provided in Table 3.

TABLE 3

Family 3 beta-glycosidases

				Identity
		GenPept		with
		Accession	SEQ ID	TrCel3A
Organism	Protein	Number	NO: 1	(%)

Aspergillus aculeatus F-50	b-glucosidase 1 (Bgl1)	BAA10968.1	72	44.1
Aspergillus fumigatus Af293	b-glucosidase	EAL91070.1	73	35.2
	(AFUA_1G17410; Afu1g17410)
Aspergillus fumigatus Af293	b-glucosidase	EAL88289.1	74	43.1
	(AFUA_1G05770; Afu1g05770)
Aspergillus kawachii ifo4308	b-glucosidase (Bg1A)	BAA19913.1	75	42.6
Aspergillus nidulans FGSC A4	b-glucosidase (AN1804.2)	EAA64969.1	76	13.2
Aspergillus nidulans FGSC A4	b-glucosidase (AN0712.2)	EAA65189.1	77	19.4
Aspergillus niger B1	b-glucosidase/tannase	CAB75696.1	78	42.8
	(Bgl1; BG3; BGs; SP188)
Aspergillus niger CBS 513.88	An18g03570 (Bgl1)	CAK48740.1	79	42.6
Aspergillus oryzae	b-glucosidase	CAD67686.1	80	42.2
Aspergillus oryzae RIB 40	b-glucosidase	BAE54829.1	81	42.3
	(AO090009000356)
Aspergillus oryzae RIB 40	b-glucosidase 5	BAE57053.1	82	43.4
	(Bgl5; AO090001000544)
Aspergillus oryzae RIB 40	b-glucosidase 1	BAE58551.1	83	31.6
	(Bgl1; AO090003001511)
Botryotinia fuckeliana	b-glucosidase (Sap1)	CAB61489.1	84	35.9
Coccidioides posadasii C735	b-glucosidase/exo-b-1,3-	AAF21242.1	85	41.4
	glucosidase (Bgl2)
Coccidioides posadasii C735	b-glucosidase (Bgl1)	AAB67972.1	86	40.4
Dictyostelium discoideum Ax3	b-glucosidase	AAA74233.1	87	16.6
Hordeum vulgare	exo-1,3-glucanase II	AAC49170.1	88	19.5
	(EII; ExoII)
Hypocrea jecorina QM6a	Cel3c - Cel3C	AAP57756.1	89	19.4
Hypocrea jecorina QM6a	Cel3e - Cel3E	AAP57760.1	90	36.5
Hypocrea jecorina QM6a	Cel3d (fragment) - Cel3D	AAP57759.1	91	12.8
Hypocrea jecorina QM6a	b-glucosidase - Cel3B	AAP57755.1	92	41.5
Hypocrea jecorina RutC-30	b-xylosidase (Bxl1)	CAA93248.1	93	19.2
Kluyveromyces fragilis	b-glucosidase	CAA29353.1	94	18.0
Kuraishia capsulata 35M5N	b-glucosidase (BglN)	AAA91297.1	95	33.0
Nicotiana tabacum	b-glucosidase	BAA33065.1	96	20.0
Periconia sp. BCC 2871	b-glucosidase	ABX84365.1	97	41.9
Phaeosphaeria avenaria WAC1293	b-glucosidase (Bgl1)	CAB82861.1	98	42.7
Phanerochaete chrysosporium K-3	glucan b-1,3-glucosidase (Bgl)	BAB85988.1	99	52.6
Phanerochaete chrysosporium	glucan	1,3-b-glucosidase (CbgL) -	AAC26489.1	100	52.6
OGC101	Bgl1A
Pichia anomala var. acetaetherius	b-glucosidase	CAA26662.1	101	36.4
Piromyces sp. E2	b-glucosidase (Cel3A) - Cel3A	AAO41704.1	102	38.3
Saccharomycopsis fibuligera	b-glucosidase 2 (Bgl2)	AAA34315.1	103	38.2
Saccharomycopsis fibuligera	b-glucosidase 1 (Bgl1)	AAA34314.1	104	38.3
Schizosaccharomyces pombe 972h-	b-glucosidase (SPBC1683.04)	CAB91166.1	105	17.4
Septoria lycopersici	b-glucosidase	AAB08445.1	106	37.2
	(tomatinase; B2Tom)
Talaromyces emersonii	b-glucosidase - Cel3A	AAL69548.3	107	43.2
Thermoascus aurantiacus	b-glucosidase (Bg2; BGII)	AAY33982.1	108	45
Thermoascus aurantiacus IFO 9748	b-glucosidase (Bgl1; Bg1)	AAZ95587.1	109	44.3
Thermoascus aurantiacus var.	b-1,4-glucosidase (Bgl1)	ABX79552.1	110	44.3
levisporus
Thermoascus aurantiacus var.	b-1,4-glucosidase (Bgl2)	ABX56926.1	111	45
levisporus
Trichoderma viride AS 3.3711	b-D-glucoside glucohydrolase	AAQ76093.1	112	98.3
	(Bgl1)
Tropaeolum majus	b-glucosidase	CAA07070.1	113	20.3
Uromyces viciae-fabae	b-glucosidase (Bgl1)	CAE01320.1	114	39.8
Volvariella volvacea V14	b-glucosidase (Bgl; BGL-II)	AAG59831.1	115	18.8

EXAMPLES

The present invention will be further illustrated in the following examples. However, it is to be understood that these examples are for illustrative purposes only and should not be used to limit the scope of the present invention in any manner.
Example 1 describes the strains and vectors used in the following examples. Example 2 describes the cloning of the TrCel3A gene and transformation in yeast. Example 3 summarizes the preparation of the error prone-PCR and site-saturation mutagenesis libraries of TrCel3A. Example 4 describes the selection and expression of wild-type and modified TrCel3A beta-glucosidases from microculture. Example 5 describes the high-throughput screening assays to identify modified TrCel3A beta-glucosidases with improved kinetic parameters. Example 6 describes the construction of modified TrCel3A beta-glucosidases comprising multiple amino acid substitutions. Example 7 describes the expression and purification of wild-type and modified TrCel3A beta-glucosidases from large scale flask cultures. Example 8 describes the measurement of the K_Gand K_G2of modified and native TrCel3A beta-glucosidases and the calculation of kinetic parameters. Example 9 describes the measurement and calculation of k_catfor wild-type and modified TrCel3A beta-glucosidases. Example 10 describes the construction of genetically modified Trichoderma reesei strains expressing cellulase mixtures comprising modified TrCel3A beta-glucosidases and the production of such cellulase mixtures in submerged liquid culture fermentation. Example 11 describes the hydrolysis of pretreated lignocellulosic substrates with cellulase mixtures comprising parental and modified TrCel3A beta-glucosidases.

Example 1

Strains and Vectors

Saccharomyces cerevisiae strain BJ3505 (pep4::HIS3 prb-Δ1.6R HIS3 lys2-208 trp1-Δ101 ura3-52 gal2 can1) was obtained from Sigma and was a part of the Amino-Terminal Yeast FLAG Expression Kit. The YEp352/PGK91-1 vector was obtained from the National Institute of Health. The pGEM T-easy vector was obtained from Promega. The vector pC/XBG1-TV is described in U.S. Pat. No. 6,105,703.

Example 2

Cloning of the TrCel3A Gene into YEp352/PGK91-1 and Transformation in Yeast

The TrCel3A gene (SEQ ID NO: 44) contains two introns. One intron is located in the secretion signal at position 323 by to 391 bp, while the other is located within the gene at position 2152 by to 2215 bp. The TrCel3A gene contains a unique NheI site located at position 1203 bp. In order to facilitate expression from yeast and cloning using NheI and KpnI restriction enzymes, the unique NheI located within TrCel3A at position 1203 by and the second intron were removed by a three step PCR. The TrCel3A gene was amplified in three segments from a plasmid containing a genomic subclone of the coding region, including introns, of the mature TrCel3A beta-glucosidase, pC/XBG1-TV using iPROOF DNA polymerase (BioRad). The first fragment (A) was amplified using primers which introduced an NheI site at the 5′ end of the gene downstream of the secretion signal (AT048) and which removed the internal NheI site (AT051). The second fragment (B) was amplified using primers which removed the internal NheI site (AT050) and the intron at position 2152 to 2215 by (AT053). The third fragment (C) was amplified using primers which removed the intron at position 2152 to 2215 by (AT052) and introduced a KpnI site at the 3′ end of the gene, downstream of the stop codon (AT049). Gene products B and C were joined together (to make gene product D) using PCR with primers AT050 and AT049. Gene product D was joined with gene product A using PCR with primers AT048 and AT049 to obtain TrCel3A without introns and with unique NheI and KpnI sites at the 5′ and 3′ ends, respectively. The final gene product was cloned into the pGEM T-easy vector (Promega) as per the manufacturer's instructions to make plasmid pGEM-TrCel3A. Primer sequences are shown below:

(SEQ ID NO: 24)

AT048: 5′ CGC CAG GCT AGC GTT GTA CCT CCT GC

(SEQ ID NO: 25)

AT049: 5′ CTG AGG GTA CCG CTA CGC TAC CGA C

(SEQ ID NO: 26)

AT050: 5′ CCC GCT AGT ATT GCC GTC GTT GGA TC

(SEQ ID NO: 27)

AT051: 5′ CCA ACG ACG GCA ATA CTA GCG GGC TTC

(SEQ ID NO: 28)

AT052: 5′ GTT CGG CTA TGG ACT GTC TTA CAC CAA GTT

CAA CTA C

(SEQ ID NO: 29)

AT053: 5′ GTT GAA CTT GGT GTA AGA CAG TCC ATA GCC

GAA CTC

Preparation of YEp352/PGK91-1/α_ssTrCel3A was conducted as follows. A DNA adapter containing NheI, KpnI, and EcoRI restriction sites was prepared by annealing primers AT046 and AT047 together. The adapter was inserted into a YEp based-plasmid containing the pgk1 promoter, alpha mating factor secretion signal, and pgk1 terminator sequences to make plasmid YEp352/PGK91-1/α_ssNKE. Specifically, the adapter was inserted as an NheI/EcoRI fragment into the NheI and EcoRI sites located downstream of the alpha mating factor secretion signal and upstream of the pgk1 terminator. Primer sequences are shown below:
(SEQ ID NO: 30)

AT046: 5′ CTA GCT GAT CAC TGA GGT ACC G

(SEQ ID NO: 31)

AT047: 5′ AAT TCG GTA CCT CAG TGA TCA G
Plasmid pGEM-TrCel3A was digested with NheI and EcoRI to release the 2235 by TrCel3A gene. The fragment was purified and ligated into the NheI and EcoRI sites of YEp352/PGK91-1/α_ssNKE to obtain YEp352/PGK91-1/α_ssTrCel3A. The resulting vector YEp352/PGK91-1/α_ss-TrCel3A was transformed in yeast strain BJ3505 using the procedure described by Gietz, R. D. and Woods, R. A. (2002).
Preparation of YEp352/PGK91-1/α _ss6H-TrCel3A was conducted as follows. A DNA adapter containing SpeI, NheI, KpnI, and EcoRI restriction sites was prepared by annealing primers AT044 and AT045 together. The adapter contains sequences coding for six histidine residues downstream of the SpeI site and upstream of the NheI site. The adapter was inserted into a YEp based-plasmid containing the pgk1 promoter, alpha mating factor secretion signal, and pgk1 terminator sequences to make plasmid YEp352/PGK91-1/α_ss6HNKE. Specifically, the adapter was inserted as an NheI/EcoRI fragment into the NheI and EcoRI sites located downstream of the alpha mating factor secretion signal and upstream of the pgk1 terminator. Primer sequences are shown below:

(SEQ ID NO: 32)

AT044: 5′ CTA GTC ATC ACC ATC ACC ATC ACG CTA GCT

GAT CAC TGA GGT ACC G

(SEQ ID NO: 33)

AT045: 5′ AAT TCG GTA CCT CAG TGA TCA GCT AGC GTG

ATG GTG ATG GTG ATG A

Plasmid pGEM-TrCel3A was digested with NheI and EcoRI to release the 2235 by TrCel3A gene. The fragment was purified and ligated into the NheI and EcoRI sites of YEp352/PGK91-1/α_ss6HNKE to obtain YEp352/PGK91-1/α _ss6H-TrCel3A. The resulting vector YEp352/PGK91-1/α _ss6H-TrCel3A (FIG. 1) was transformed in yeast strain BJ3505 using the procedure described by Gietz, R. D. and Woods, R. A. (2002).

Example 3

Random Mutagenesis of TrCel3A

a. Error Prone-PCR
A random mutagenesis library was generated by error-prone PCR using a Mutazyme® II DNA polymerase method. A series of four independent PCRs was performed using 5, 10, 15, 20 η_gof YEp352/PGK91-1/α _ss6H-TrCel3A vector and the Mutazyme® II DNA polymerase with primers YαN21 and 3′PGK-term. Annealing temperature was set to 50° C. The amplification was done for 20 cycles. The four PCR products were pooled and diluted to 16 ηg/μL. The YEp352/PGK91-1/α _ss6H-TrCel3A vector was digested with NheI and KpnI and the empty vector fragment was isolated. This linear fragment and the final amplicon were transformed simultaneously and cloned by in vivo recombination into yeast strain BJ3505 (Butler, T. and Alcalde, M., 2003).
(SEQ ID NO: 34)

YαN21: 5′AGC ACA AAT AAC GGG TTA TTG

(SEQ ID NO: 35)

3′PGK-term: 5′GCA ACA CCT GGC AAT TCC TTA CC

b. Site-Saturation Mutagenesis
Four TrCel3A libraries were created using site-saturation mutagenesis (SSM) with degenerate primers (NNS) targeting amino acid positions V43, V101, F260, and I543. SSM was performed using a two-step PCR method involving megaprimer synthesis followed by PCR-mediated overlap extension. PCR reactions were carried out using the High Fidelity iProof Taq Polymerase (BioRad). YEp352/PGK91-1/α _ss6H-TrCel3A was used as the template for the V43X, F260X, and I543X libraries, while YEp352/PGK91-1/α _ss6H-TrCel3A (S72N, F96L, V101M, N369K, A386T) served as the template for the V101X library.
For each SSM library, MegaPrimer A was amplified using the external primer YαN21 with an internal reverse primer, while MegaPrimer B was derived by combining the external primer PGKterm with an internal forward primer. The internal forward primers contained a degenerate codon sequence to introduce random amino acid substitutions within their target sites. The megaprimers were purified using the Wizard® SV Gel and PCR Clean-Up System.

(SEQ ID NO: 34)

YαN21: 5′AGC ACA AAT AAC GGG TTA TTG

(SEQ ID NO: 35)

PGKterm: 5′GCA ACA CCT GGC CCT TAC C

(SEQ ID NO: 36)

V43X-F: 5′TGG AAC GGC GGT CCT TGC NNS GGA AAC ACA

TCT CCG GCC TC

(SEQ ID NO: 37)

V43X-R: 5′GCA AGG ACC GCC GTT CCA

(SEQ ID NO: 38)

M101X-F: 5′C GGT GAG GAG NNS AAG GCC TCG G

(SEQ ID NO: 39)

M101X-R: 5′ATG AAC TGT CCA CGT TCG CGG

(SEQ ID NO: 40)

F260X-F: 5′TG TCA ATG CCT GGC ACA GAC NNS AAC GGT

AAC AAT CGG

(SEQ ID NO: 41)

F260X-R: 5′GT CTG TGC CAG GCA TTG ACA

(SEQ ID NO: 42)

I543X-F: 5′CCC AAT GAC TAT AAC ACT CGC NNS GTT TCC

GGC GGC AGT GAC

(SEQ ID NO: 43)

I543X-R: 5′GCG AGT GTT ATA GTC ATT GGG

In the second round of PCR, both megaprimers for a given SSM library were allowed to anneal and extend for 10 cycles to generate the final template. The external primers YαN21 and PGKterm were then added for another 25 cycles to amplify the final product, which was subsequently purified using the Wizard® SV Gel and PCR Clean-Up System. Both the purified PCR product and the linearized vector YEp352/PGK91-1α_ss-6H-TrCel3A (NheI+NruI) were transformed and cloned via in vivo recombination within the BJ3505 yeast strain using the procedure described by Gietz, R. D. and Woods (2002).

Example 4

Selection and Growth of Yeast Strains Expressing Parental and Modified TrCel3A Beta-Glucosidases

This example describes the selection and expression of parental (TrCel3A^Wt) and modified TrCel3A beta-glucosidase from Saccharomyces cerevisiae for use in high-throughput screening assays.
S. cerevisiae transformants were grown on plates containing synthetic complete medium (SC: 2% agar w/v, 0.17% yeast nitrogen base w/v, 0.192%-Ura drop-out supplement w/v, 2% glucose w/v, 2% casamino acids w/v, 0.5% ammonium sulfate w/v, pH 5.5) for 3-4 days at 30° C. Each growth plate was replicated by transferring a portion of each colony, using sterilized velvet, to a screen-out plate containing SC medium plus 0.1% esculin hydrate and 0.03% FeCl₃. Colonies which turned black after incubation for 3-4 days at 30° C. were identified as expressing active beta-glucosidase (FIG. 2). Colonies were correlated back to their original growth plate and selected for liquid media expression cultures by toothpick inoculation of 1 mL SC media in sterile 96-deepwell plates containing one glass bead (1.5-2.0 mm diameter). Expression cultures were grown for 3 days at 30° C. and 245 rpm with humidity control (New Brunswick Scientific Innova®44 incubator shaker series). Glycerol stocks were prepared by transferring 50 μL of liquid culture to the corresponding wells of a microplate containing 50 μL of 40% glycerol and stored at −80° C. Expression culture plates were centrifuged at 1600×g for 5 minutes to pellet cells and supernatant was aspirated for screening assays (Example 5).

Example 5

Screening of TrCel3A Gene Libraries for Modified TrCel3A Beta-Glucosidases with Higher Catalytic Efficiency

This example describes the screening of modified TrCel3A beta-glucosidases for increased higher catalytic efficiency by comparison to parental TrCel3A that had been cloned into Saccharomyces cerevisiae.
Modified TrCel3A beta-glucosidases expressed from yeast as described in Example 4 were tested in two 80 μL citrate buffered (pH 5) cellobiose hydrolysis assays using a 96-well PCR plate format. A 40 μL aliquot of supernatant containing a parental or modified TrCel3A beta-glucosidase was incubated with 30 mM cellobiose (Assay 1) and 5.0 mM cellobiose plus 1.25 mM glucose (Assay 2) for 5, 10, 20 and 40 min at 50° C. in an MJ Research Tetrad™2 Peltier Thermal Cycler. Contained in each 96-well PCR plate were six parental TrCel3A controls used for comparison. Enzyme activity was measured through the detection of glucose using a glucose oxidase-peroxidase coupled assay (Trinder P., 1969). Exogenous glucose (1.25 mM) included in Assay 2 was subtracted from the total amount of glucose measured following the incubation. An Assay 2/Assay 1 enzyme activity ratio was calculated for the parental (TrCel3A^Wtor Wt) and all modified TrCel3A beta-glucosidases by dividing the enzyme activity in Assay 2 by the enzyme activity in Assay 1. The Assay 2/Assay 1 activity ratio for each modified TrCel3A beta-glucosidase was then compared to that of the average of six parental TrCel3A beta-glucosidase controls on a particular microplate and positives were selected at the 95% confidence level using a t-test. All positive modified TrCel3A beta-glucosidases were produced again in microculture and re-screened to reduce the number of false positives (FIG. 3). Table 4 lists the positive modified TrCel3A beta-glucosidases obtained from screening the error-prone and site-saturation libraries (Example 3) and the Assay 2/Assay 1 enzyme activity ratios compared to the parental, wild-type TrCel3A beta-glucosidase.

TABLE 4

Modified TrCel3A beta-glucosidases selected random libraries.

		Assay 2/Assay 1 Enzyme Activity Ratio
	Modified TrCel3A	(normalized to TrCel3A^Wt)

	Wt	1.00
	V43I	1.20
	V43C	1.36
	V101A	1.17
	V101G	1.17
	F260I	1.14
	I543N	1.12
	I543W	1.26
	I543A	1.23
	I543G	1.22
	I543L	1.21
	I543S	1.25

Example 6

Construction and Expression of Modified TrCel3A Beta-Glucosidases with Multiple Amino Acid Substitutions

Using YEp352/PGK91-1/α _ss6H-TrCel3A(S72N-F96L-V101M (U.S. Publication No. 2010/0093040A1) as a template, additional mutations were introduced using a two-step PCR method involving megaprimer synthesis followed by megaprimer PCR using High Fidelity iProof Taq Polymerase (Table 5). The internal primers were modified to introduce the desired amino acid substitutions into the TrCel3A construct. The external plasmid primers (YαN21 and PGKterm) were used to amplify the final product. The megaprimers and final products were purified using the Wizard® SV Gel and PCR Clean-Up System.

TABLE 5

Generation of modified TrCel3A enzymes by PCR.

PCR	Step	Template	Primer 1	Primer 2	Amplicon

1	1	YEp352/PGK91-1-α_SS-6H-	YαN21	DK068	PCR 1 Step 1
		TrCel3A(S72N-F96L-V101M)
	1	YEp352/PGK91-1-α_SS-6H-	DK067	PGK-Term	PCR 1 Step 1
		TrCel3A(S72N-F96L-V101M)
	2	Both PCR 1 Step 1	YαN21	PGK-Term	TrCel3A(S72N-V101M)
		megaprimers
2	1	TrCel3A(S72N-V101M)	YαN21	DK222	PCR 2 Step 1
	1	TrCel3A(S72N-V101M)	DK221	PGK-Term	PCR 2 Step 1
	2	Both PCR 2 Step 1	YαN21	PGK-Term	TrCel3A(V43I-S72N-V101M)
		megaprimers
3	1	TrCel3A(S72N-V101M)	YαN21	DK106	PCR 3 Step 1
	1	TrCel3A(S72N-V101M)	DK105	PGK-Term	PCR 3 Step 1
	2	Both PCR 3 Step 1	YαN21	PGK-Term	TrCel3A(S72N-V101M-
		megaprimers			F260I)
4	1	TrCel3A(S72N-V101M)	YαN21	DK224	PCR 4 Step 1
	1	TrCel3A(S72N-V101M)	DK223	PGK-Term	PCR 4 Step 1
	2	Both PCR 4 Step 1	YαN21	PGK-Term	TrCel3A(S72N-V101M-
		megaprimers			I543D)
5	1	TrCel3A(S72N-V101M)	YαN21	DK232	PCR 5 Step 1
	1	TrCel3A(S72N-V101M)	DK231	PGK-Term	PCR 5 Step 1
	2	Both PCR 5 Step 1	YαN21	PGK-Term	TrCel3A(S72N-V101M-I543L)
		megaprimers
6	1	TrCel3A(S72N-V101M)	YαN21	DK230	PCR 6 Step 1
	1	TrCel3A(S72N-V101M)	DK229	PGK-Term	PCR 6 Step 1
	2	Both PCR 6 Step 1	YαN21	PGK-Term	TrCel3A(S72N-V101M-
		megaprimers			I543N)
7	1	TrCel3A(V43I-S72N-V101M)	YαN21	DK230	PCR 7 Step 1
	1	TrCel3A(V43I-S72N-V101M)	DK229	PGK-Term	PCR 7 Step 1
	2	Both PCR 7 Step 1	YαN21	PGK-Term	TrCel3A(V43I-S72N-V101M-
		megaprimers			I543N)
8	1	TrCel3A(S72N-V101M-F260I)	YαN21	DK222	PCR 8 Step 1
	1	TrCel3A(S72N-V101M-F260I)	DK221	PGK-Term	PCR 8 Step 1
	2	Both PCR 8 Step 1	YαN21	PGK-Term	TrCel3A(V43I-S72N-V101M-
		megaprimers			F260I)
9	1	TrCel3A(S72N-V101M-F260I)	YαN21	DK232	PCR 9 Step 1
	1	TrCel3A(S72N-V101M-F260I)	DK231	PGK-Term	PCR 9 Step 1
	2	Both PCR 9 Step 1	YαN21	PGK-Term	TrCel3A(S72N-V101M-
		megaprimers			F260I-I543L)
10	1	TrCel3A(S72N-V101M-F260I)	YαN21	DK230	PCR 10 Step 1
	1	TrCel3A(S72N-V101M-F260I)	DK229	PGK-Term	PCR 10 Step 1
	2	Both PCR 10 Step 1	YαN21	PGK-Term	TrCel3A(S72N-V101M-
		megaprimers			F260I-I543N)
11	1	TrCel3A(S72N-V101M-F260I-	YαN21	DK222	PCR 11 Step 1
		I543N)
	1	TrCel3A(S72N-V101M-F260I-I543N)	DK221	PGK-Term	PCR 11 Step 1
	2	Both PCR 11 Step 1	YαN21	PGK-Term	TrCel3A(V43I-S72N-V101M-
		megaprimers			F260I-I543N)

To facilitate cloning, the final product was digested with NheI+KpnI and ligated into vector YEp352/PGK91-1/α _ss6H-TrCel3A linearized with NheI+KpnI. The ligation mix was transformed into DH5a chemically-competent E. coli cells, plasmid extracted, and sequenced. Plasmids encoding the modified beta-glucosidases were transformed into yeast strain BJ3505.
(SEQ ID NO: 34)

YαN21 5′-AGCACAAATAACGGGTTATTG-3′

(SEQ ID NO: 35)

3′PGKterm 5′-GCAACACCTGGCCCTTACC-3′

(SEQ ID NO: 45)

5′DK067 5′-CGCGAACGTGGACAGTTCATCGGTGAGGAGATG-3′

(SEQ ID NO: 46)

3′DK068 5′-CATCTCCTCACCGATGAACTGTCCACGTTCGCG-3′

(SEQ ID NO: 47)

5′DK105 5′-CAATGCCTGGCACAGACATCAACGGTAACAATC-3′

(SEQ ID NO: 48)

3′DK106 5′-GATTGTTACCGTTGATGTCTGTGCCAGGCATTG-3′

(SEQ ID NO: 49)

5′DK221 5′-GGCGGTCCTTGCATTGGAAACACAT-3′

(SEQ ID NO: 50)

3′DK222 5′-ATGTGTTTCCAATGCAAGGACCGCC-3′

(SEQ ID NO: 51)

5′DK223 5′-GACTATAACACTCGCGACGTTTCCGGCGGCAG-3′

(SEQ ID NO: 52)

3′DK224 5′-CTGCCGCCGGAAACGTCGCGAGTGTTATAGTC-3′

(SEQ ID NO: 53)

5′DK229 5′-GACTATAACACTCGCAACGTTTCCGGCGGCAG-3′

(SEQ ID NO: 54)

3′DK230 5′-CTGCCGCCGGAAACGTTGCGAGTGTTATAGTC-3′

(SEQ ID NO: 55)

5′DK231 5′-GACTATAACACTCGCCTGGTTTCCGGCGGCAG-3′

(SEQ ID NO: 56)

3′DK232 5′-CTGCCGCCGGAAACCAGGCGAGTGTTATAGTC-3′

Example 7

Purification of Modified TrCel3A Beta-Glucosidases

Modified TrCel3A beta-glucosidases that passed the selection criteria in Example 5, along with the modified TrCel3A-F260X beta-glucosidases produced by site-saturation mutagenesis at position 260 (Example 4) or by combining two or more amino acid substitutions (Example 6), were purified for further analysis. For each modified TrCel3A beta-glucosidase, 50 mL of sterile YPD medium (10 g/L yeast extract, 20 g/L peptone and 20 g/L glucose) was inoculated with 10 mL of overnight cultures of transformed Saccharomyces cerevisiae grown from cells freshly picked from an agar plate. The cultures were then incubated for 96 hours at 30° C. with shaking at 200 rpm.
After incubation, the broth from each culture was centrifuged for 10 minutes at 9000 rpm and the pellet (containing yeast cells) discarded. The pH of the supernatant was adjusted to 5.0. The TrCel3A in each spent culture medium was then purified by immobilized metal affinity chromatography (IMAC) using His-Trap NTA/Ni²⁺ columns from GE Healthcare (catalogue #17-5247-01). Purified proteins were concentrated and buffer exchanged using Vivaspin 20 centrifugal concentrators (Sartorius Stedim Biotech, catalogue No. VS2012). Protein concentrations were measured using the method of Bradford (1976) and stored at −20° C. Samples of each purified Modified TrCel3A were separated by SDS-PAGE and visualized by Coomassie Blue stain (FIG. 4).

Example 8

Measuring the K_Gand K_G2Constants of Parental and Modified TrCel3A Beta-Glucosidases

The K_Gand K_G2constants of each modified TrCel3A beta-glucosidase were determined using a p-nitrophenyl-beta-D-glucopyranoside (pNPG) competitive substrate/inhibitor real-time kinetic assay. Each modified TrCel3A (3 μg/reaction) was incubated with 0.4 mM pNPG in a stirred cuvette; the total reaction volume was 3 mL. Assays were buffered using 50 mM citrate, pH 5.0. Incubations were done at 50° C. for up to 40 min in a Varian Cary UV/Vis spectrophotometer. Absorbance measurements collected at 340 nm during the time course were converted to p-nitrophenol (pNP) concentration using Equation 1.
$\begin{matrix} pNP = \frac{{Abs}_{340 nm} - ɛ_{p} NPG \cdot pNPG}{ɛ_{p} NP - ɛ_{p} NPG} & Equation 1 \end{matrix}$
where,
pNP is the concentration of p-nitrophenol (mM),
Abs_340nmis the absorbance at 340 nm,
ε_pNpGis the extinction coefficient at 340 nm of pNPG at pH 5.0 (3.33 L cm⁻¹±0.04)
pNPG is the initial concentration of pNPG (mM),
ε_pNPis the extinction coefficient at 340 nm of pNP at pH 5.0 (5.96 L cm⁻¹±0.02)
Three different incubations were done for each modified TrCel3A: 1) with pNPG alone, 2) with pNPG and 3 mM cellobiose, and 3) with pNPG and 5 mM glucose. The pNP concentration as a function of time in each of the three reactions was modeled according to Equation 2 using a fourth order Runge-Kutta workbook in MS Excel and using the method of least squares.
$\begin{matrix} \frac{\partial pNP}{\partial t} = \frac{k_{pNPG} \cdot E \cdot pNPG}{pNPG + K_{pNPG} (1 + \frac{G 2}{K_{G 2}} + \frac{G}{K_{G}})} & Equation 2 \end{matrix}$
where,
dpNP/dt is the rate of conversion of pNPG to pNP (mM/min),
k_pNPGis the catalytic rate constant for the conversion of pNPG to pNP (μmol/min/mg protein),
E is the concentration of TrCel3A (mg/mL),
pNPG is the concentration of p-nitrophenyl-beta-D-glucopyranoside (mM),
K_pNPGis the Michaelis constant (or K_m) for pNPG (mM),
G2 is the concentration of cellobiose (mM),
K_G2is the Michaelis constant (or K_m) for cellobiose (mM),
G is the concentration of glucose (mM),
K_Gis the glucose inhibition constant (mM).
The reaction scheme for this assay is shown in FIG. 5. In this model, Cel3A hydrolyzes pNPG according to Michaelis-Menten kinetics. Cel3A activity is assumed to be inhibited competitively by glucose as described by the inhibition constant, K_G. Therefore, when glucose was added to a cuvette containing pNPG, the rate of pNPG catalysis decreased. The decrease in the rate of pNPG hydrolysis is accounted for by the K_Gparameter. Modified TrCel3A beta-glucosidases with a higher K_Gvalue are less affected by glucose, compared to the parental TrCel3A^Wt, and will have relatively higher rates of pNPG hydrolysis under these conditions. Similarly, when cellobiose was included in the reaction with pNPG, the rate of pNPG hydrolysis decreased. Modified TrCel3A beta-glucosidases with a lower K_G2value are more affected by the addition of cellobiose, compared to TrCel3A^Wt, and will have relatively lower rates of pNPG hydrolysis under these conditions.
The rates of pNPG hydrolysis were assayed for each modified TrCel3A in each of the three conditions, pNPG alone, pNPG+G2 and pNPG+G, by using a global fit of these three data sets to the parameters k_pNPG, K_pNPG, k_cat, K_G2and K_Gin manner known by one of skill in the art. A K_G/K_G2ratio was also calculated using the values of K_Gand K_G2from each global fit of the three data sets for each modified TrCel3A.
Each modified TrCel3A was assayed in this manner between two and five times. The average K_G, K_G2and K_G/K_G2values determined in this manner and their standard deviations are shown in Table 6. Student's t-test was used to identify modified TrCel3A beta-glucosidases with statistically significant improvements in K_G, K_G2and K_G/K_G2(P<0.05) compared to TrCel3A^wt. Graphs showing representative pNPG hydrolysis data and model fits for TrCel3A-WT (FIG. 6), TrCel3A-V43I (FIG. 7), TrCel3A-V101A (FIG. 8), TrCel3A-F260I (FIG. 9) and TrCel3A-I543N (FIG. 10) are also shown.
The K_Gvalues of TrCel3A-V43C (4.20 mM), TrCel3A-F260I (0.92 mM), TrCel3A-F260D (0.70 mM), TrCel3A-F260Q (0.69 mM), TrCel3A-F260V (0.72 mM) and TrCel3A-I543N (0.94 mM) were higher than that of TrCel3A^Wt(0.58 mM). This indicates that the activity of each of these modified TrCel3A beta-glucosidases is significantly less inhibited by glucose and that they maintain relatively higher activity in the presence of glucose than does the parental TrCel3A beta-glucosidase. The values of K_G2of several modified TrCel3As with single amino acid substitutions, such as TrCel3A-V43I (0.62 mM) and TrCel3A-V101A (0.82 mM), were significantly (<0.001 and 0.001, respectively) lower than the K_G2of wild-type TrCel3A (1.18 mM). Similarly, most of the modified TrCel3As with different combinations of more than one amino acid substitution had lower K_G2than wild-type. These modified TrCel3A beta-glucosidases exhibit maximum activity at lower concentrations of cellobiose, indicating that they have a higher affinity for cellobiose. In a cellulose hydrolysis system, such as the conversion of cellulose to fermentable sugars utilizing cellulase such as that from Trichoderma reesei or other cellulolytic fungi, the use of a TrCel3A with a lower K_G2would contribute to lowering steady-state concentrations of cellobiose and lower product inhibition of cellulase enzymes.
The value of K_G, K_G2, K_G/K_G2and k_G2for each modified TrCel3A was divided by the value of the corresponding parameter for the parental TrCel3A in order to calculate the relative values shown in Table 2. These results show that the TrCel3A-V43I (1.41), TrCel3A-V101A (1.34), TrCel3A-F260I (1.52) and TrCel3A-I543N (1.35) beta-glucosidases have substantially improved K_G/K_G2; improvements ranged from 35-52%, relative to TrCel3A^Wt.

Example 9

Measuring the Catalytic Rate Constant of Parental and Modified TrCel3A Beta-Glucosidases

Initial rate assays were used to measure the catalytic rate constant (k_cat) of the parental and each modified TrCel3A beta-glucosidase on cellobiose. Purified wild-type parental and modified TrCel3A beta-glucosidases were incubated with 12 concentrations of cellobiose, ranging from 0.3 to 40 mM. The protein concentration in each of the reactions was 1 μg/mL. Samples were incubated at 50° C. for 15 min in deep well plates and then placed in a boiling water bath for 10 min to stop the reaction. The concentration of glucose produced at each concentration of substrate was measured as described in Example 7.
The rate of cellobiose consumption for the parental and each modified TrCel3A beta-glucosidase was plotted as a function of cellobiose concentration. As the cellobiose concentration increases from 0.4 mM to 10 mM, the reaction rate of wild-type TrCel3A increases until it reaches an apparent maximum reaction rate (FIG. 12). Further increasing the substrate concentration results in a gradual decrease in the reaction rate, a phenomenon that is reportedly due to substrate inhibition (Cascalheira et al., 1999). As a result, data for the reaction rate as a function of cellobiose concentration were modeled using a modified form of the Michaelis-Menten equation which incorporates a K_siterm for uncompetitive substrate inhibition (Equation 3).
$\begin{matrix} \frac{\partial G 2}{\partial t} = \frac{\frac{k_{cat} \cdot E \cdot G 2}{K_{G 2}}}{1 + \frac{G 2}{K_{G 2}} + \frac{G 2^{2}}{K_{G 2} \cdot K_{si}}} & Equation 3 \end{matrix}$
where,
dG2/dt is the rate of conversion of cellobiose (G2) to two glucose molecules (2G) (mM G2 consumed/min),
k_catis the catalytic rate constant for the conversion of cellobiose to glucose (limo' of G2 consumed/min/mg protein),
E is the concentration of TrCel3A (mg/mL),
G2 is the concentration of cellobiose (mM),
K_G2is the Michaelis constant (or Km) for cellobiose (mM),
K_Siis the cellobiose substrate inhibition constant (mM)
The k_catis the TrCel3A rate constant on cellobiose and K_Siis the parameter that describes the substrate inhibition. The data for the parental and each modified TrCel3A beta-glucosidase were fit to this model by non-linear regression using the method of least squares as known to those of skill in the art. The parental and each modified TrCel3A beta-glucosidase were assayed in triplicate on three different occasions. The mean values of k_cat, K_siand their standard deviations are shown in Table 6.
The k_catof several modified TrCel3As, including TrCel3A-F260I (11.06 mmol/min/mg), TrCel3A-I543S (9.76 μmol/min/mg) and TrCel3A-I543L (11.50 μmol/min/mg) was significantly higher than that of wild-type TrCel3A (8.92 μmol/min/mg) (Table 6 and FIG. 12). Therefore, these modified beta-glucosidases catalyze the conversion of cellobiose to two glucose molecules at a faster rate than does wild-type TrCel3A. In a cellulose hydrolysis system, such as the conversion of cellulose to fermentable sugars utilizing cellulase such as that from Trichoderma reesei, the use of a TrCel3A with a higher k_catwould contribute to lowering steady-state concentrations of cellobiose and lower product inhibition of cellulase enzymes.

TABLE 6

Kinetic Parameters of Parental and Modified TrCel3A beta-glucosidases.

	K_G	P-value	K_G2	P-value		P-value	k_cat	P-value	K_si
TrCel3A-	(mM)	(K_G)	(mM)	(K_G2)	K_G/K_G2	(K_G/K_G2)	(μmol/min/mg)	(k_cat)	(mM)

V43I	0.42 ± 0.04	<0.001	0.65 ± 0.07	<0.001	0.66 ± 0.06	<0.001	6.73 ± 0.13	0.018	64.1
V43C	4.20 ± 0.57	<0.001	2.39 ± 0.33	<0.001	1.79 ± 0.38	<0.001	ND^a	—	ND^a
V101A	0.54 ± 0.18	0.593	0.82 ± 0.16	0.001	0.65 ± 0.09	0.006	7.22 ± 0.18	0.049	52.5
F260I	0.92 ± 0.04	<0.001	1.23 ± 0.03	0.22	0.75 ± 0.02	<0.001	11.06 ± 0.29	0.017	60.8
F260D	0.70	—	1.31	—	0.53	—	ND^a	—	ND^a
F260Q	0.69	—	1.34	—	0.51	—	ND^a	—	ND^a
F260V	0.72	—	1.28	—	0.56	—	ND^a	—	ND^a
I543N	0.87 ± 0.12	0.001	1.39 ± 0.21	0.064	0.64 ± 0.11	0.019	6.53 ± 0.17	0.006	85.5
I543W	0.56 ± 0.08	0.635	0.95 ± 0.08	0.003	0.59 ± 0.06	0.041	4.53 ± 0.24	<0.01	42.3
1543S	0.63	—	1.08	—	0.59	—	9.76 ± 0.15	<0.01	102.7
I543A	0.68	—	1.16	—	0.59	—	8.96 ± 0.19	0.944	94.5
I543G	0.54	—	1.02	—	0.53	—	8.02 ± 0.14	<0.01	98.7
I543L	0.61	—	1.13	—	0.55	—	11.50 ± 0.12	<0.01	129.4
S72N-V101M-	0.44 ± 0.03	<0.001	0.68 ± 0.04	<0.001	0.65 ± 0.05	0.019	8.45 ± 0.35	0.605	38.3
F260I
V43I-S72N-	0.34 ± 0.08	0.001	0.46 ± 0.04	<0.001	0.73 ± 0.04	<0.001	7.55 ± 0.19	0.034	38.0
V101M
S72N-V101M-	0.43 ± 0.05	0.001	0.64 ± 0.04	<0.001	0.66 ± 0.03	<0.001	7.73 ± 0.13	<0.01	40.8
I543N
S72N-V101M-	0.36 ± 0.02	<0.001	0.53 ± 0.04	<0.001	0.68 ± 0.03	0.005	ND		ND
I543D
S72N-V101M-	0.42 ± 0.04	<0.001	0.69 ± 0.06	<0.001	0.61 ± 0.06	0.004	8.57 ± 0.30	0.638	35.0
I543L
V43I-S72N-	0.34 ± 0.09	0.009	0.36 ± 0.09	<0.001	0.93 ± 0.08	<0.001	6.98 ± 0.25	0.029	34.6
V101M-F260I
S72N-V101M-	0.64 ± 0.04	0.108	0.84 ± 0.04	<0.001	0.77 ± 0.08	0.004	9.12 ± 0.14	0.580	31.8
F260I-I543N
V43I-S72N-	0.41 ± 0.02	<0.001	0.38 ± 0.04	<0.001	1.10 ± 0.11	0.001	6.14 ± 0.04	<0.001	49.3
V101M-
F260I-I543N
V43I-S72N-	0.30 ± 0.01	<0.001	0.32 ± 0.03	<0.001	0.95 ± 0.09	0.001	6.51 ± 0.07	<0.001	51.7
V101M-I543N
S72N-V101M-	0.50 ± 0.03	0.010	0.66 ± 0.06	<0.001	0.77 ± 0.10	0.008	8.88 ± 0.24	0.816	53.6
F260I-I543L
Wt	0.58 ± 0.09	—	1.18 ± 0.12	—	0.49 ± 0.06	—	8.92 ± 0.29	—	66.1
(parental)

The K_G, K_G2, K_G/K_G2, k_catand K_Siparameters for parental (TrCel3A-Wt) and modified beta-glucosidases were determined as described in Examples 8 and 9. Significant improvements (P value < 0.05) in any of these parameters are shown in bold text.
^aND = not determined

Example 10

Expression of Modified Cel3A in Trichoderma reesei

10.1: Trichoderma reesei Strains
Strain P59G is a genetically modified strain that produces and secretes high levels of the beta-glucosidase encoded by T. reesei bgl1 as described in U.S. Pat. No. 6,015,703. The parent strain of P59G and modified Cel3A over-expressing transformant 4115A, is strain BTR213aux. The strain BTR213 is a derivative of RutC30 (ATCC #56765; Montenecourt and Eveleigh, 1979) produced by random mutagenesis and first selected for ability to produce larger clearing zones on minimal media agar containing 1% acid swollen cellulose and 4 g L⁻¹2-deoxyglucose and then selected for the ability to grow on lactose media containing 0.2 μg/mL carbendazim. A uridine auxotroph of BTR213, BTR213aux, was obtained through selection of mutants spontaneously resistant to 0.15% w/v 5-fluoroorotic-acid (FOA).
10.2: Generation of T. reesei Transformation Vector
The T. reesei expression vector was generated using pUC19 vector (Fermentas) as a backbone. To introduce spacers and cloning sites required for cloning of selection and expression cassettes two DNA fragments were amplified using pCAMBIA1301 plasmid (see URL: cambia.org/daisy/cambia/materials/vectors.525.html#dsy585) as a template and two pairs of primers AC166/AC167 and AC168/AC169 (Table 7). The first fragment was cloned into the EcoRI/SacI sites of pUC19 introducing two new PacI/AflII restriction sites. The second fragment was cloned into the SacI/BamHI sites introducing NotI/MluI restriction sites and generating pUC19-GDR vector.
For the construction of the TrCel3A expression cassette, a fragment containing the TrCel7A promoter and xylanase 2 secretion signal (Pcel7a-Xyn2ss fragment) was amplified using primers AC230/AC231 (Table 7) and pC/XBG-TV vector (U.S. Pat. No. 6,015,703) as template. The gene encoding TrCel3A-S72N-V101M-F260I (described in Example 6) was amplified using primers AC232 and AC233 (Table 7). The Pcel7a-Xyn2ss fragment was ligated to the TrCel3A-572N-V101M-F260I encoding gene in two subsequent PCR reactions using primers AC230 and AC233 (Table 7) to produce the resulting c/xCel3A-572N-V101M-F2601 fragment. A fragment comprising the cel6a terminator (Tcel6a fragment) was amplified from the pC/XBG-TV (U.S. Pat. No. 6,015,703) template using primers Tcel6a-F and Tcel6a-R (Table 7), which introduced BamHI/KpnI restriction sites, respectively. The c/xCel3A-572N-V101M-F2601 and Tcel6a fragments were cloned into pGEM-Teasy vectors generating vectors pGEM-c/xCel3A-572N-V101M-F2601 and pGEM-Tce16a which were then digested with MluI/KpnI and BamHI/KpnI restriction enzymes to release the c/xCel3A-572N-V101M-F2601 and Tce16a fragments, respectively. Both fragments were gel isolated and cloned by three fragment ligation into MluI/BamHI sites of the pUC-GDR vector, generating pc/xCel3A-572N-V101M-F260I. To eliminate the SbfI restriction site, pc/xCel3A-572N-V101M-F2601 was digested with XbaI/SphI and the ends modified by removal of the 5′ overhang and filling in of the 3′ overhang. The linear plasmid was then ligated back together generating pc/xCel3A-572N-V101M-F260I-Sbf. Next, a 2.2 kb fragment containing the N. crassa orotidine-5′-phosphate decarboxylase (pyr4) gene was amplified from pNcBgl (U.S. Pat. No. 6,939,704) containing N. crassa pyr4 gene under control of its native promoter and terminator using primers AC323 and AC343 (Table 7). The pyr4 cassette was cloned into pGEM-T-easy, digested with PacI/NotI restriction enzymes, gel purified and cloned into PacI/NotI sites of pc/xCel3A-S72N-V101M-F260I-Sbf generating final transformation vector pc/xCel3A-S72N-V101M-F260I-pyr4-TV (FIG. 13).

TABLE 7

Primers used for construction of T. reesei transformation
vector pc/xCel3A-AT012; F260I-pyr4-TV.

Primer name	Primer sequence

AC166	ACTGAATTCTTAATTAAGAACCGACGACTCGTCCGTC (SEQ ID NO: 57)

AC167	GTGGAGCTCCTTAAGGTGACATCGGCTTCAAATGGC (SEQ ID NO: 58)

AC168	GCAGAGCTCGCGGCCGCGAACCGACGACTCGTCCGTC (SEQ ID NO: 59)

AC169	CTGGGATCCGATATCACGCGTGTGACATCGGCTTCAAATGGC (SEQ ID NO:
	60)

AC230	TTTACGCGTGATTATGGCGTACTAGAGAGCGG (SEQ ID NO: 61)

AC231	CTGCAGGAGGTACAACCTGGCGCTTCTCCACAGCCACGG (SEQ ID NO: 62)

AC232	GTGGAGAAGCGCCAGGTTGTACCTCCTGCAGGGACTCCATG (SEQ ID NO: 63)

AC233	TTTGGTACCCTACGCTACCGACAGAGTGCTCG (SEQ ID NO: 64)

AC323	TTTGCGGCCGCCATCATTCGTCGCTTTCGG (SEQ ID NO: 65)

AC343	TTCGATCGACTATACCACCACCCACCG (SEQ ID NO: 66)

Tcel6A-F	CTGGGTACCGGCTTTCGTGACCGGGCTTC (SEQ ID NO: 67)

Tcel6A-R	CTGGGATCCGATGGACTAGTACAGCCATG (SEQ ID NO: 68)

10.3: Trichoderma reesei Transformation and Characterization of Transformants

Trichoderma strain 4115A was generated by transformation of pc/xCel3A-S72N-V101M-F260I-pyr4-TV into strain BTR213aux by biolistic gold particle bombardment using the PDS-1000/He system (BioRad; E.I. DuPont de Nemours and Company). Gold particles (median diameter of 0.6 um, BioRad Cat. No. 1652262) were used as microcarriers. The following parameters were used in the optimization of the transformation: a rupture pressure of 1100 psi, a helium pressure of 29 mm Hg, a gap distance of 6 mm and a target distance of 6 cm. The spore suspension was prepared by washing T. reesei spores from PDA plates incubated for 4-5 days at 30° C. with sterile water. About 5×10⁷spores were plated on 60 mm diameter plates containing minimal media agar (MM). After particle delivery, all transformation plates were incubated at 30° C. for 5-10 days. All transformants were transferred to minimal media agar and incubated at 30° C.

Minimal medium (MM*) agar:

	Component	Amount for 1 L of medium

KH₂PO₄	10	g
(NH₄)₂SO₄	6	g
Na₃Citrate•2H₂O	3	g
FeSO₄•7H₂O	5	mg
MnSO₄•H₂O	1.6	mg
ZnSO₄•7H₂O	1.4	mg
CaCl₂•2H₂O	2	mg
Agar	20	g
20% Glucose f.s.	50	ml
1 M MgSO₄•7H₂O f.s.	4	mL

	pH to 5.5

	*MMSS agar contains the same components as MM agar plus 1.2 M sorbitol, 4 mM MgSO₄, 1 g/L YNB (Yeast Nitrogen Base w/o Amino Acids from DIFCO Cat. No. 291940) and 0.12 g/L amino acids (-Ura DO Supplement from CLONTECH Cat. No. 8601-1).

All T. reesei transformants were pre-screened for production of active modified TrCel3A beta-glucosidase using Esculin (β-D-glucose-6,7-dihydroxycoumarin) plate assay. An esculin stock solution was made by mixing 2 g of Esculin and 0.6 g FeCl₃in 200 mL of deionized water. The mixture was heated until dissolved, cooled to approximately 40° C. and filter sterilized. Transformants were plated on minimal media agar plates containing 1% cellobiose and grown for 3 to 4 days at 30° C. The esculin stock solution was diluted four-fold with 250 mM citrate buffer, pH 4.8 and 15 mL of the diluted esculin solution were overlaid onto the plates. Plates were incubated at 30° C. for one hour. Positive transformants were selected according to formation of black precipitate formed around T. reesei colonies.
Individual Trichoderma colonies selected for the expression of active modified TrCel3A beta-glucosidases were transferred to potato dextrose agar (PDA) (Difco) plates and allowed to sporulate. At that time, about 10⁴-10⁶spores of each individual transformant, parental and P59G strains were used to inoculate 1 mL of microculture medium in 24-well micro-plates.

Microculture medium

	Component	g/L

	(NH₄)₂SO₄	12.7
	KH₂PO₄	8.0
	MgSO₄•7H₂O	4.0
	CaCl₂•2H₂O	1.02
	CSL	5.0
	CaCO₃	20.0
	Carbon source**	30-35
	Trace elements*	2 mL/L
		pH 5.5

	*Trace elements solution contains 5 g/L FeSO₄•7H ₂0; 1.6 g/L MnSO₄•H ₂0; 1.4 g/Ll ZnSO₄•7H ₂0.
	**glucose, Solka floc, lactose, cellobiose, sophorose, corn syrup, or Avicel. The carbon source can be sterilized separately as an aqueous solution at pH 2 to 7 and added to the remaining media initially or through the course of the fermentation.

The cultures were incubated at a temperature of 30° C. with shaking at 250 rpm for 6 days. The biomass was separated from growth media containing the secreted protein by centrifugation at 12000 rpm. The protein concentration was determined using the Bio-Rad Protein Assay (Cat. No. 500-0001).
The concentration of Cel3A in supernatants from Trichoderma reesei microcultures was determined by ELISA (FIG. 14). Filtrate and purified component standard were diluted 0.01-10 μg/mL (based on total protein) in phosphate-buffered saline, pH 7.2 (PBS) and incubated overnight at 4° C. in microtitre plates (Costar EIA #9018). These plates were washed with PBS containing 0.1% Tween-20 (PBS/Tween) and then incubated in PBS containing 1% bovine serum albumin (PBS/BSA) for 1 hr at room temperature. Blocked microtitre wells were washed with PBS/Tween. Rabbit polyclonal antisera specific for TrCel3A was diluted (1:8,000) in PBS/BSA, added to separate microtitre plates and incubated for 2 h at room temperature. Plates were washed and incubated with a goat anti-rabbit antibody coupled to horseradish peroxidase (Sigma #A6154), diluted 1/2000 in PBS/BSA, for 1 h at room temperature. After washing, tetramethylbenzidine was added to each plate and incubated for 30 min at room temperature. The absorbance at 360 nm was measured in each well and converted into protein concentration using a TrCel3A standard curve.
10.4: Trichoderma reesei Pilot Fermentation
For 14 L pilot fermentations, T. reesei strains were grown on Potato Dextrose Agar at 28-30° C. until a confluent lawn of spores was obtained. Spores were collected and used to inoculate 750 ml of Berkeley media (10 g/L glucose, 1.4 g/L (NH₄)₂SO₄, 2.0 g/L KH₂PO₄, 0.31 g/L MgSO₄.7H₂O, 0.53 g/L CaCl₂, 5.1 g/L dry corn steep, 5 mg/L FeSO₄.7H₂O, 0.8 mg/L MnSO₄.H₂O, 0.7 mg/L ZnSO₄.7H₂O) in a 2 L baffled flask. After 3 days of growth at 28° C. and 150 rpm, this culture was used to inoculate 10 L of fermentation medium with the following initial composition: 13 g/l glucose, 2.2 g/l (NH₄)₂SO₄, 1.39 g/l KH₂PO₄, 0.7 g/l MgSO₄.7H₂O, 0.185 g/l CaCl₂, 6 g/l dry corn steep, 1.75 mg/l FeSO₄.7H₂O, 0.56 mg/l MnSO₄.H₂O, 0.49 g/l ZnSO₄.7H₂O. A fed-batch aerobic fermentation using an inducing carbohydrate source was run for 6 days at pH 5 and 28° C. in a 14 L New Brunswick Microferm fermentor. After 6 days, the culture was filtered over Harborlite 1500S and the culture filtrate saved.
The concentration of parental or modified TrCel3A beta-glucosidases in fermentation filtrate from Trichoderma reesei was determined by ELISA as described above (Example 10.3).

Example 11

Measuring the Cellulose Hydrolysis Activity of a Whole Cellulase Secreted by a Strain of Trichoderma that Expresses High Levels of a Modified Beta-Glucosidase

A whole enzyme produced by a strain of Trichoderma that expresses high concentrations of a modified beta-glucosidase, TrCel3A-S72N-V101M-F260I, was compared to that from a strain that expresses similar concentrations of the parental, wild-type TrCel3A in an extended hydrolysis time course assay on a lignocellulosic substrate. The concentrations of the wild-type and the modified beta-glucosidase in their respective whole cellulase mixtures were 31.1±1.7% and 28.2±1.3% of total protein, respectively. Whole Trichoderma cellulase mixtures containing these TrCel3A beta-glucosidases were incubated with pretreated wheat straw at a concentration of 25 g/L cellulose at a dose of 10 mg of total cellulase mixture per gram of cellulose. Triplicate assays were performed for each cellulase mixture under the same conditions. The hydrolysis assays were buffered in 50 mM citrate, pH 5.0 containing 0.1% sodium benzoate. The assay was conducted at 50° C. for 96 hr with continuous orbital shaking. Aliquots of 0.7 mL were taken at various time points from each flask and the glucose concentration in the soluble portion was assayed and converted into a measure of fractional cellulose conversion. The conversion data were then fit with a rectangular hyperbola with an additional linear term using minimization of the sum of squared residuals of fit. The equation was of the following form: conversion=(max*time)/(halfmax+time)+c*time. Both sets of data were fit globally with unique max and halfmax values and a shared value of the variable c.
The results are shown in FIG. 15. This figure demonstrates that the fractional cellulose conversion measured for the cellulase mixture containing the modified TrCel3A beta-glucosidase was higher at 96 hr (0.99±0.03) than for the cellulase mixture containing the wild-type TrCel3A beta-glucosidase (0.89±0.02). This increase in fractional cellulose conversion was statistically significant (P<0.05, Student's T-Test).

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Claims

1. A modified Trichoderma reesei TrCel3A beta-glucosidase comprising one or more amino acid substitutions selected from the group consisting of V43X, V101X, F260X and I543X, the modified TrCel3A beta-glucosidase comprising an amino acid sequence which is from about 80% to about 99.9% identical to SEQ ID NO: 1.

2. The modified Trichoderma reesei TrCel3A beta-glucosidase of claim 1, wherein the modified Trichoderma reesei TrCel3A beta-glucosidase exhibits

a. at least a 20% increase in K_G,

b. at least a 20% decrease in K_G2, or

c. at least a 10% increase in k_cat

relative to the K_G, K_G2, or k_catof a parental Trichoderma reesei TrCel3A from which the modified Trichoderma reesei TrCel3A is derived.

3. The modified Trichoderma reesei TrCel3A beta-glucosidase of claim 2, wherein the modified Trichoderma reesei TrCel3A beta-glucosidase exhibits

a. at least a 30% increase in K_G,

b. at least a 30% decrease in K_G2, or

c. at least a 20% increase in k_cat

4. The modified Trichoderma reesei TrCel3A beta-glucosidase of claim 1, comprising an amino acid sequence which from about 90% to about 99.9% identical to SEQ ID NO: 1.

5. The modified Trichoderma reesei TrCel3A beta-glucosidase of claim 1, wherein the one or more of the amino acid substitutions is selected from the group consisting of V43I, V43C, V101A, V101G, F260I, F260V, F260Q, F260D, I543N, I543W, I543A, I543S, I543G, and I543L.

6. The modified Trichoderma reesei TrCel3A beta-glucosidase of claim 2, wherein the one or more of the amino acid substitutions is selected from the group consisting of V43I, V43C, V101A, V101G, F260I, F260V, F260Q, F260D, I543N, I543W, I543A, I543S, I543G, and I543L.

7. The modified Trichoderma reesei TrCel3A beta-glucosidase of claim 5, wherein the modified Trichoderma reesei TrCel3A beta-glucosidase exhibits

a. at least a 20% increase in K_G,

b. at least a 20% decrease in K_G2, or

c. at least a 10% increase in k_cat

relative to a parental Trichoderma reesei TrCel3A from which the modified Trichoderma reesei TrCel3A is derived.

8. The modified Trichoderma reesei TrCel3A beta-glucosidase of claim 7, wherein the modified Trichoderma reesei TrCel3A beta-glucosidase exhibits

a. at least a 30% increase in K_G,

b. at least a 30% decrease in K_G2, or

c. at least a 20% increase in k_cat

9. The modified Trichoderma reesei TrCel3A beta-glucosidase of claim 1, further comprising one or more amino acid substitutions selected from the group consisting of V66X, S72X, F96X, T235X, N248X, N369X and A386X.

10. The modified Trichoderma reesei TrCel3A beta-glucosidase of claim 2, further comprising one or more amino acid substitutions selected from the group consisting of V66X, S72X, F96X, T235X, N248X, N369X and A386X.

11. The modified Trichoderma reesei TrCel3A beta-glucosidase of claim 5, further comprising one or more amino acid substitutions selected from the group consisting of V66X, S72X, F96X, T235X, N248X, N369X and A386X.

12. An isolated genetic construct comprising a nucleic acid sequence encoding a modified Trichoderma reesei TrCel3A beta-glucosidase comprising one or more of the amino acid substitutions selected from the group consisting of V43X, V101X, F260X and I543X, wherein the amino acid sequence of the modified TrCel3A beta-glucosidase encoded by said construct comprises a sequence which is from about 80% to about 99.9% identical to SEQ ID NO: 1.

13. An isolated genetic construct comprising DNA encoding a modified Trichoderma reesei TrCel3A beta-glucosidase comprising one or more of the amino acid substitutions selected from the group consisting of V43X, V101X, F260X and I543X, wherein the amino acid sequence of the modified TrCel3A beta-glucosidase encoded by said construct is from about 80% to about 99.9% identical to SEQ ID NO: 1 and the modified Trichoderma reesei TrCel3A beta-glucosidase exhibits

a. at least a 20% increase in K_G,

b. at least a 20% decrease in K_G2, or

c. at least a 10% increase in k_cat

14. An isolated genetically modified microbe comprising the genetic construct of claim 12.

15. An isolated genetically modified microbe comprising the genetic construct of claim 13.

16. The isolated genetically modified microbe of claim 14, wherein said microbe is a species of yeast or filamentous fungus.

17. The isolated genetically modified microbe of claim 15, wherein said microbe is a species of yeast or filamentous fungus.

18. The isolated genetically modified microbe of claim 16, wherein said microbe is Saccharomyces cerevisiae or Trichoderma reesei.

19. The isolated genetically modified microbe of claim 17, wherein said microbe is Saccharomyces cerevisiae or Trichoderma reesei.

20. A process for producing a modified Trichoderma reesei TrCel3A beta-glucosidase, comprising the steps of (i) providing a genetically modified microbe comprising the genetic construct of claim 12 (ii) culturing the genetically modified microbe in submerged liquid fermentations under conditions that induce the expression of the modified Trichoderma reesei TrCel3A beta-glucosidase, and (iii) recovering the modified Trichoderma reesei TrCel3A beta-glucosidase.

21. A process for producing a modified Trichoderma reesei TrCel3A beta-glucosidase, comprising the steps of (i) providing a genetically modified microbe comprising the genetic construct of claim 13 (ii) culturing the genetically modified microbe in submerged liquid fermentations under conditions that induce the expression of the modified Trichoderma reesei TrCel3A beta-glucosidase, and (iii) recovering the modified Trichoderma reesei TrCel3A beta-glucosidase.

22. A process for enzymatic hydrolysis of a cellulose substrate comprising contacting said substrate with one or more cellulase enzymes and the modified Trichoderma reesei TrCel3A beta-glucosidase of claim 1.

23. A process for enzymatic hydrolysis of a cellulose substrate comprising contacting said substrate with one or more cellulase enzymes and the modified Trichoderma reesei TrCel3A beta-glucosidase of claim 2.

24. The process of claim 22, wherein the cellulose substrate is a pretreated lignocellulosic feedstock and wherein the process produces fermentable sugars.

25. The process of claim 23, wherein the cellulose substrate is a pretreated lignocellulosic feedstock and wherein the process produces fermentable sugars.

26. The process of claim 24, wherein the pretreated lignocellulose feedstock is selected from the group consisting of corn stover, wheat straw, barley straw, rice straw, oat straw, canola straw, sugarcane straw, soybean stover, corn fiber, sugar beet pulp, pulp mill fines and rejects, sugar cane bagasse, hardwood, softwood, sawdust, switch grass, miscanthus, cord grass, and reed canary grass.

27. The process of claim 25, wherein the pretreated lignocellulose feedstock is selected from the group consisting of corn stover, wheat straw, barley straw, rice straw, oat straw, canola straw, sugarcane straw, soybean stover, corn fiber, sugar beet pulp, pulp mill fines and rejects, sugar cane bagasse, hardwood, softwood, sawdust, switch grass, miscanthus, cord grass, and reed canary grass.

28. A modified Trichoderma reesei TrCel3A beta-glucosidase comprising the amino acid sequence of one of:

SEQ ID NO: 2 (TrCel3A-V43I);

SEQ ID NO: 3 (TrCel3A0V43C);

SEQ ID NO: 4 (TrCel3A-V101A);

SEQ ID NO: 5 (TrCel3A-V101G);

SEQ ID NO: 6 (TrCel3A-F260I);

SEQ ID NO: 7 (TrCel3A-F260V);

SEQ ID NO: 8 (TrCel3A-F260Q);

SEQ ID NO: 9 (TrCel3A-F260D);

SEQ ID NO: 10 (TrCel3A-I543N);

SEQ ID NO: 11 (TrCel3A-I543W);

SEQ ID NO: 12 (TrCel3A-I543A);

SEQ ID NO: 13 (TrCel3A-I543S);

SEQ ID NO: 14 (TrCel3A-I543G);

SEQ ID NO: 15 (TrCel3A-I543L);

SEQ ID NO: 16 (TrCel3A-572N-V101M-F260I);

SEQ ID NO: 17 (TrCel3A-V43I-572N-V101M);

SEQ ID NO: 18 (TrCel3A-572N-V101M-I543N);

SEQ ID NO: 19 (TrCel3A-572N-V101M-I543D);

SEQ ID NO: 20 (TrCel3A-572N-V101M-I543L);

SEQ ID NO: 21 (TrCel3A-V43′-572N-V101M-F260I);

SEQ ID NO: 22 (TrCel3A-V43′-572N-V101M-F260I-I543N);

SEQ ID NO: 23 (TrCel3A-V43I-572E-V101M-I543N);

SEQ ID NO: 69 (TrCel3A-572N-V101M-F260I-I543N); or

SEQ ID NO: 70 (TrCel3A-572N-V101M-F260I-I543L).

29. A modified Family 3 beta-glycosidase comprising one or more of the amino acid substitutions selected from the group consisting of selected from the group consisting of V43I, V43C, V101A, V101G, F260I, F260V, F260Q, F260D, I543N, I543W, I543A, I543S, I543G, and I543L, said position determined from alignment of a parental Family 3 beta-glycosidase with the TrCel3A amino acid sequence as defined in SEQ ID NO: 1, wherein the amino acid sequence of the modified Family 3 beta-glycosidase comprises a sequence which is from about 80% to about 99.9% identical to an amino acid sequence of a parental Family 3 beta-glycosidase from which the modified Family 3 beta-glycosidase is derived.

30. The modified Family 3 beta-glycosidase of claim 29, wherein the amino acid sequence of the modified Family 3 beta-glycosidase is from about 80% to about 99.9% identical to an amino acid sequence of a parental Family 3 beta-glycosidase from which the modified Family 3 beta-glycosidase is derived, the modified Family 3 beta-glycosidase exhibiting

a. an increase in K_P,

b. a decrease in K_S, or

c. an increase in k_cat

relative to the K_P, K_S, or k_catof a parental Family 3 beta-glycosidase from which the modified Family 3 beta-glycosidase is derived.